Mycoplasma hyopneumoniae vaccine

ABSTRACT

This invention provides an immunogenic composition including a soluble portion of a  Mycoplasma hyopneumoniae  (M.hyo) whole cell preparation, wherein the soluble portion of the M.hyo preparation is substantially free of both (i) IgG and (ii) immunocomplexes comprised of antigen bound to immunoglobulin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/850,318, filed Mar. 26, 2013, which claims the benefit of U.S.Provisional Application No. 61/620,165, filed Apr. 4, 2012, the contentseach of which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to Mycoplasma hyopneumoniae (M.hyopneumoniae or M.hyo). More particularly, the invention relates to thesoluble portion of an M.hyo whole cell preparation and its use in avaccine for protecting pigs against enzootic pneumonia.

BACKGROUND OF THE INVENTION

Enzootic pneumonia in swine, also called mycoplasmal pneumonia, iscaused by M.hyo. The disease is a chronic, non-fatal disease affectingpigs of all ages. Infected pigs show only mild symptoms of coughs andfever, but the disease has significant economic impact due to reducedfeed efficiency and reduced weight gain. Enzootic pneumonia istransmitted from pig to pig through the nasal passages by airborneorganisms expelled from the lungs of infected pigs. The primaryinfection by M.hyo may be followed by secondary infection by othermycoplasma species (Mycoplasma hyorhinis and Mycoplasma flocculare) aswell as other bacterial pathogens.

M.hyo is a small, prokaryotic microbe capable of a free livingexistence, although it is often found in association with eukaryoticcells because it has absolute requirements for exogenous sterols andfatty acids. These requirements generally necessitate growth inserum-containing media. M.hyo is bounded by a cell membrane, but not acell wall.

The physical association of mycoplasmas with the host cell surface isthe basis for the development and persistence of enzootic pneumonia.M.hyo infects the respiratory tract of swine, colonizing the trachea,bronchi, and bronchioles. The mycoplasma produces a ciliostatic factorwhich causes the cilia lining the respiratory passages to stop beating.Eventually, the cilia degenerate, leaving the pig prone to infection bysecondary pathogens. Characteristic lesions of purple to gray areas ofconsolidation are observed in infected animals. Surveys of slaughteredanimals revealed lesions in 30 to 80% of swine. Results from 37 herds in13 states indicated that 99% of the herds had hogs with pneumonialesions typical of enzootic pneumonia. Therefore, the need for effectivepreventative and treatment measures are great.

Antibiotics such as tiamulin, trimethoprim, tetracyclines and lincomycinhave some benefit, but are expensive and require prolonged use.Additionally, antibiotics have not been shown to effectively eliminatespread or reinfection of M.hyo. Prevention by maintaining pathogen-freeherds is sometimes possible but reintroduction of M.hyo often occurs.Due to the serious economic consequences of swine pneumonia, vaccinesagainst M.hyo have been sought. Vaccines containing preparations ofmycoplasmal organisms grown in serum-containing medium have beenmarketed, but raise concerns regarding adverse reactions induced byserum components (such as immunocomplexes or non-immunogenic specificproteins) present in the immunizing material. Other attempts to provideM. hyo vaccines have been successful, but the disease remainswidespread.

M.hyo and porcine circovirus type 2 (PCV2) are the two most prevalentpathogens that are encountered in the pig industry. Swine infected withPCV2 exhibit a syndrome commonly referred to as Post-weaningMultisystemic Wasting Syndrome (PMWS). PMWS is clinically characterizedby wasting, paleness of the skin, unthriftiness, respiratory distress,diarrhea, icterus, and jaundice. In addition to PMWS, PCV2 has beenassociated with several other infections including pseudorabies, porcinereproductive and respiratory syndrome (PRRS), Glasser's disease,streptococcal meningitis, salmonellosis, postweaning colibacillosis,dietetic hepatosis, and suppurative bronchopneumonia. M.hyo isassociated with enzootic pneumonia and has also been implicated as oneof the major co-factors in the development of Porcine CircovirusAssociated Disease (PCVAD).

Porcine reproductive and respiratory syndrome (PRRS) is caused by anarterivirus, which has a particular affinity for the macrophagesparticularly those found in the lung (alveolar macrophages). Thesemacrophages ingest and remove invading bacteria and viruses, but not inthe case of the PRRS virus. In the case of the PRRS virus, it multipliesinside the macrophages producing more virus and kills the macrophages.Once PRRSV has entered a herd, it tends to remain present and activeindefinitely. Up to 40% of the macrophages are destroyed, which allowsbacteria and other viruses to proliferate and do damage. A commonexample of this is the noticeable increase in severity of enzooticpneumonia in grower/finisher units when they become infected with PRRSvirus. More than half of weaning-age PRRS virus-negative pigs becomeinfected before going to market.

What is needed is an improved vaccine against mycoplasma infection inswine. Preferably, the M.hyo vaccine will be compatible with otherporcine antigens, such as PCV2 and PRRS virus, whether they are givenconcurrently as separate single vaccines or combined in a ready-to-usevaccine. It would be highly desirable to provide a ready-to-use,single-dose M.hyo/PCV2 combination vaccine.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic composition including asoluble portion of an Mycoplasma hyopneumoniae (M.hyo) whole cellpreparation, wherein the soluble portion of the M.hyo preparation issubstantially free of both (i) IgG and (ii) immunocomplexes comprised ofantigen bound to immunoglobulin. In one aspect, the soluble portion ofthe M.hyo whole cell preparation has been treated with protein-A orprotein-G prior to being added to the immunogenic composition.

In one embodiment, the soluble portion of the M.hyo preparation includesat least one M.hyo protein antigen. In another embodiment, the solubleportion of the M.hyo preparation includes two or more M.hyo proteinantigens.

In some embodiments, the immunogenic composition of the presentinvention further includes at least one additional antigen. In oneembodiment, the at least one additional antigen is protective against amicroorganism that can cause disease in pigs.

In one embodiment, the microorganism includes bacteria, viruses, orprotozoans. In another embodiment, the microorganism is selected from,but is not limited to, the following: porcine circovirus type 2 (PCV2),porcine reproductive and respiratory syndrome virus (PRRSV), porcineparvovirus (PPV), Haemophilus parasuis, Pasteurella multocida,Streptococcum suis, Staphylococcus hyicus, Actinobacillluspleuropneumoniae, Bordetella bronchiseptica, Salmonella choleraesuis,Salmonella enteritidis, Erysipelothrix rhusiopathiae, Mycoplamahyorhinis, Mycoplasma hyosynoviae, leptospira bacteria, Lawsoniaintracellularis, swine influenza virus (SIV), Escherichia coli antigen,Brachyspira hyodysenteriae, porcine respiratory coronavirus, PorcineEpidemic Diarrhea (PED) virus, rotavirus, Torque teno virus (TTV),Porcine Cytomegalovirus, Porcine enteroviruses, Encephalomyocarditisvirus, a pathogen causative of Aujesky's Disease, Classical Swine fever(CSF) and a pathogen causative of Swine Transmissable Gastroenteritis,or combinations thereof.

In certain embodiments, the at least one additional antigen is a porcinecircovirus type 2 (PCV2) antigen, a PRRS virus antigen or a combinationthereof. In one embodiment, the composition elicits a protective immuneresponse in a pig against both M.hyo and PCV2. In another embodiment,the composition elicits a protective immune response in a pig againstM.hyo, PCV2 and PRRS virus.

In one embodiment, the PCV2 antigen is in the form of a chimerictype-1-type 2 circovirus, the chimeric virus including an inactivatedrecombinant porcine circovirus type 1 expressing the porcine circovirustype 2 ORF2 protein. In another embodiment, the PCV2 antigen is in theform of a recombinant ORF2 protein. In still another embodiment, therecombinant ORF2 protein is expressed from a baculovirus vector.

In some embodiments, the composition of the present invention furtherincludes an adjuvant. In one embodiment, the adjuvant is selected from,but is not limited to, the following: an oil-in-water adjuvant, apolymer and water adjuvant, a water-in-oil adjuvant, an aluminumhydroxide adjuvant, a vitamin E adjuvant and combinations thereof. Inanother embodiment, the composition of the present invention furtherincludes a pharmaceutically acceptable carrier.

In certain embodiments, the composition of the present invention elicitsa protective immune response against M.hyo when administered as a singledose administration. In further embodiments, the composition elicits aprotective immune response against M.hyo and at least one additionalmicroorganism that can cause disease in pigs when administered as asingle dose administration. In still further embodiments, a compositionof the present invention elicits a protective response against bothM.hyo and at least one additional microorganism that causes disease inpigs when administered as a two dose administration.

The present invention also provides a method of immunizing a pig againstM.hyo. This method includes administering to the pig an immunogeniccomposition including a soluble portion of an M.hyo whole cellpreparation, wherein the soluble portion of the M.hyo preparation issubstantially free of both (i) IgG and (ii) immunocomplexes comprised ofantigen bound to immunoglobulin. In one embodiment, the soluble portionof the M.hyo preparation of the administered composition includes atleast one M.hyo protein antigen.

In one embodiment of the method of the present invention, thecomposition is administered intramuscularly, intradermally,transdermally, or subcutaneously. In another embodiment of the method ofthis invention, the composition is administered in a single dose. In yetanother embodiment of the method of this invention, the composition isadministered as two doses.

In a further embodiment of the method of the present invention, thecomposition is administered in conjunction with at least one additionalantigen that is protective against a microorganism that can causedisease in pigs, such as one or more of the microorganisms describedabove. Such other antigens can be given concurrently with the M.hyocomposition (i.e., as separate single vaccines) or combined in aready-to-use vaccine.

In a further embodiment, the composition is administered to pigs havingmaternally derived antibodies against M.hyo. In a still furtherembodiment, the composition is administered to pigs having maternallyderived antibodies against both M.hyo and at least one othermicroorganism that can cause disease in pigs.

In one embodiment, the composition is administered to pigs at 3 weeks ofage or older.

The present invention further provides a kit. This kit includes a bottlecomprising an immunogenic composition. This immunogenic compositionincludes the soluble portion of a Mycoplasma hyopneumoniae (M.hyo) wholecell preparation, wherein the soluble portion of the M.hyo preparationis substantially free of both (i) IgG and (ii) antigen/immunoglobulinimmunocomplexes. In one embodiment, the kit further includes aninstruction manual containing information to administer the immunogeniccomposition.

Additionally, the present invention provides a method for preparing animmunogenic composition according to this invention. This methodincludes i) culturing M.hyo in a suitable media over periods rangingfrom 18-144 hours; ii) subsequently inactivating the M. hyo culture;iii) harvesting the inactivated culture fluid, wherein the inactivatedculture fluid comprises an M.hyo whole cell preparation comprising botha soluble liquid fraction and insoluble cellular material; iv)separating the soluble liquid fraction from the insoluble cellularmaterial; and v) substantially removing both IgG andantigen/immunoglobulin immunocomplexes from the separated soluble liquidfraction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the efficacy of M.hyo monovalent vaccinesprepared with M.hyo antigens from different treatments (T02-T10described in Example 3) vs. a placebo (T01). The results are presentedas % Lung Lesion Least Square Mean values.

FIG. 2 is a graph showing the PCV2 antigen potency results (PCV2 antigenELISA) of M.hyo vaccines in combination with killed PCV Type1-Type2chimeric virus. The chimeric virus was included in the compositions atan initial level of about 1.6≦RP. The status of each sample is expressedas relative potency (RP).

FIG. 3 is a graph showing the PCV2 viremia results (PCV2 QuantitativePCR) observed with PCV/M.hyo vaccine formulations employing differentadjuvant platforms.

FIG. 4 is a graph showing the PCV2 antibody ELISA (S/P) serologicalresults observed with PCV/M.hyo vaccine formulations employing differentadjuvant platforms on days 1, 20, and 42 of challenge.

FIG. 5 is a graph showing the PCV2 fecal shed obtained with the T02-T04treatments described in Example 7 vs. a placebo (T01). The results areexpressed as PCV2 DNA copies/ml.

FIG. 6 is a graph showing the PCV2 nasal shed obtained with the T02-T04treatments described in Example 7 vs. the placebo (T01). The results areexpressed as PCV2 DNA copies/ml.

FIG. 7A and FIG. 7B are graphs showing the results of aninterferon-gamma (IFN-γ) test that measures PCV2-specific cellularmediated immune (CMI) responses. The results ofpos-vaccination/pre-challenge are presented in FIG. 7A, and the resultsof post-vaccination/post-challenge are presented in FIG. 7B. Stimulationof 5×10⁶ cells was considered significant.

FIG. 8A and FIG. 8B depict the M.hyo efficacy of the PCV2/M.hyoexperimental vaccine formulations in SP-oil. The lung scores forformulations employing M.hyo treatments T02-T08 vs. a placebo (T01) aredepicted graphically in FIG. 8A. The table in FIG. 8B depicts thecontrast of treatments T02-T08 with the placebo.

FIG. 9 is a flowchart which shows one embodiment of a manufacturingprocess used to prepare PCV2-compatible Protein-A treated M.hyo antigen.

FIG. 10 is a table showing the adjuvant evaluation for virucidalactivity against PRRS virus.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is one embodiment of a nucleotide sequence encoding p46from the P-5722 strain of M.hyo;

SEQ ID NO: 2 is one embodiment of an amino acid sequence correspondingto p46 from the P-5722 strain of M.hyo;

SEQ ID NO: 3 is one embodiment of a nucleotide sequence encoding p97from the P-5722 strain of M.hyo;

SEQ ID NO: 4 is one embodiment of an amino acid sequence correspondingto p97 from the P-5722 strain of M.hyo;

SEQ ID NO: 5 is one embodiment of a genomic sequence encoding a chimericPCV1-2 virus;

SEQ ID NO: 6 is one embodiment of a nucleotide sequence corresponding toORF2 of a porcine circovirus;

SEQ ID NO: 7 is one embodiment of an amino acid sequence correspondingto the ORF2 polypeptide of a porcine circovirus;

SEQ ID NO: 8 is one embodiment of a genomic sequence encoding a chimericPCV1-2 virus;

SEQ ID NO: 9 is one embodiment of a nucleotide sequence corresponding toORF2 of a porcine circovirus;

SEQ ID NO: 10 is one embodiment of an amino acid sequence correspondingto the ORF2 polypeptide of a porcine circovirus;

SEQ ID NO: 11 is one embodiment of an amino acid sequence correspondingto the ORF2 polypeptide of a porcine circovirus;

SEQ ID NO: 12 is one embodiment of a nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 11;

SEQ ID NO: 13 is one embodiment of an amino acid sequence correspondingto the ORF2 polypeptide of a porcine circovirus;

SEQ ID NO: 14 is one embodiment of a nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 13;

SEQ ID NO: 15 is one embodiment of an amino acid sequence correspondingto the ORF2 polypeptide of a porcine circovirus;

SEQ ID NO: 16 is one embodiment of a genomic sequence of a non-virulentform of the North American PRRS virus isolate designated P 129; and

SEQ ID NO: 17 is one embodiment of a nucleotide sequence correspondingto ORF2 to ORF5 of the PRRS virus isolate designated ISU-55.

SEQ ID NO: 18 is one embodiment of a nucleotide sequence correspondingto ORF6 and ORF7 of the PRRS virus isolate designated ISU-55.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an immunogenic composition including asoluble portion of an M.hyo whole cell preparation, wherein the solubleportion of the M.hyo preparation is substantially free of both (i) IgGand (ii) antigen-bound immunocomplexes. Applicants have surprisinglydiscovered that the insoluble fraction of the M.hyo whole cellpreparation is non-immunogenic. In contrast, the IgG-free M.hyo solublepreparation is immunogenic and can be effectively combined with antigensfrom other pathogens, such as PCV2, without analytical or immunologicalinterference between the antigens. This makes the M.hyo solublepreparation of this invention an effective platform for multivalentvaccines, including one-bottle, ready-to-use formulations. Applicantshave also surprisingly discovered that removing the immunoglobulin andthe insoluble cell debris enhances the safety of the immunogeniccomposition.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a protein antigen” includes aplurality of protein antigens, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude other elements.

As defined herein, a soluble portion of an M.hyo whole cell preparationrefers to a soluble liquid fraction of an M.hyo whole cell preparationafter separation of the insoluble material and substantial removal ofIgG and antigen-bound immunocomplexes. The M.hyo soluble portion mayalternatively be referred to herein as the supernatant fraction, culturesupernatant and the like. It includes M.hyo-expressed soluble proteins(M.hyo protein antigens) that have been separated or isolated frominsoluble proteins, whole bacteria, and other insoluble M.hyo cellularmaterial by conventional means, such as centrifugation, filtration, orprecipitation. In addition to including M.hyo-specific soluble proteins,the soluble portion of the M.hyo whole cell preparation also includesheterologous proteins, such as those contained in the culture mediumused for M.hyo fermentation.

The term “antigen” refers to a compound, composition, or immunogenicsubstance that can stimulate the production of antibodies or a T-cellresponse, or both, in an animal, including compositions that areinjected or absorbed into an animal. The immune response may begenerated to the whole molecule, or to a portion of the molecule (e.g.,an epitope or hapten).

As defined herein, an “immunogenic or immunological composition”, refersto a composition of matter that comprises at least one antigen whichelicits an immunological response in the host of a cellular and orantibody-mediated immune response to the composition or vaccine ofinterest.

The term “immune response” as used herein refers to a response elicitedin an animal. An immune response may refer to cellular immunity (CMI);humoral immunity or may involve both. The present invention alsocontemplates a response limited to a part of the immune system. Usually,an “immunological response” includes, but is not limited to, one or moreof the following effects: the production or activation of antibodies, Bcells, helper T cells, suppressor T cells, and/or cytotoxic T cellsand/or yd T cells, directed specifically to an antigen or antigensincluded in the composition or vaccine of interest. Preferably, the hostwill display either a therapeutic or protective immunological responsesuch that resistance to new infection will be enhanced and/or theclinical severity of the disease reduced. Such protection will bedemonstrated by either a reduction or lack of symptoms normallydisplayed by an infected host, a quicker recovery time and/or a loweredviral titer in the infected host.

As used herein, the term “immunogenicity” means capable of producing animmune response in a host animal against an antigen or antigens. Thisimmune response forms the basis of the protective immunity elicited by avaccine against a specific infectious organism.

An “adjuvant” as used herein means a composition comprised of one ormore substances that enhances the immune response to an antigen(s). Themechanism of how an adjuvant operates is not entirely known. Someadjuvants are believed to enhance the immune response by slowlyreleasing the antigen, while other adjuvants are strongly immunogenic intheir own right and are believed to function synergistically.

As used herein, the term “multivalent” means a vaccine containing morethan one antigen whether from the same species (i.e., different isolatesof Mycoplasma hyopneumoniae), from a different species (i.e., isolatesfrom both Pasteurella hemolytica and Pasteurella multocida), or avaccine containing a combination of antigens from different genera (forexample, a vaccine comprising antigens from Pasteurella multocida,Salmonella, Escherichia coli, Haemophilus somnus and Clostridium).

The term “pig” or “piglet” as used herein means an animal of porcineorigin, while “sow” refers to a female of reproductive age andcapability. A “gilt” is a female pig who has never been pregnant.

As used herein, the term “virulent” means an isolate that retains itsability to be infectious in an animal host.

“Inactivated vaccine” means a vaccine composition containing aninfectious organism or pathogen that is no longer capable of replicationor growth. The pathogen may be bacterial, viral, protozoal or fungal inorigin. Inactivation may be accomplished by a variety of methodsincluding freeze-thawing, chemical treatment (for example, treatmentwith thimerosal or formalin), sonication, radiation, heat or any otherconvention means sufficient to prevent replication or growth of theorganism while maintaining its immunogenicity.

The term “variant” as used herein refers to a polypeptide or a nucleicacid sequence encoding a polypeptide, that has one or more conservativeamino acid variations or other minor modifications such that thecorresponding polypeptide has substantially equivalent function whencompared to the wild-type polypeptide.

“Conservative variation” denotes the replacement of an amino acidresidue by another biologically similar residue, or the replacement of anucleotide in a nucleic acid sequence such that the encoded amino acidresidue does not change or is another biologically similar residue.Examples of conservative variations include the substitution of onehydrophobic residue, such as isoleucine, valine, leucine or methioninefor another hydrophobic residue, or the substitution of one polarresidue, such as the substitution of arginine for lysine, glutamic acidfor aspartic acid, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

As used herein, the terms “pharmaceutically acceptable carrier” and“pharmaceutically acceptable vehicle” are interchangeable and refer to afluid vehicle for containing vaccine antigens that can be injected intoa host without adverse effects. Suitable pharmaceutically acceptablecarriers known in the art include, but are not limited to, sterilewater, saline, glucose, dextrose, or buffered solutions. Carriers mayinclude auxiliary agents including, but not limited to, diluents,stabilizers (i.e., sugars and amino acids), preservatives, wettingagents, emulsifying agents, pH buffering agents, viscosity enhancingadditives, colors and the like.

As used herein, the term “vaccine composition” includes at least oneantigen or immunogen in a pharmaceutically acceptable vehicle useful forinducing an immune response in a host. Vaccine compositions can beadministered in dosages and by techniques well known to those skilled inthe medical or veterinary arts, taking into consideration such factorsas the age, sex, weight, species and condition of the recipient animal,and the route of administration. The route of administration can bepercutaneous, via mucosal administration (e.g., oral, nasal, anal,vaginal) or via a parenteral route (intradermal, transdermal,intramuscular, subcutaneous, intravenous, or intraperitoneal). Vaccinecompositions can be administered alone, or can be co-administered orsequentially administered with other treatments or therapies. Forms ofadministration may include suspensions, syrups or elixirs, andpreparations for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration (e.g., injectable administration) such assterile suspensions or emulsions. Vaccine compositions may beadministered as a spray or mixed in food and/or water or delivered inadmixture with a suitable carrier, diluent, or excipient such as sterilewater, physiological saline, glucose, or the like. The compositions cancontain auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, adjuvants, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standardpharmaceutical texts, such as “Remington's Pharmaceutical Sciences,”1990 may be consulted to prepare suitable preparations, without undueexperimentation.

“North American PRRS virus” means any PRRS virus having geneticcharacteristics associated with a North American PRRS virus isolate,such as, but not limited to the PRRS virus that was first isolated inthe United States around the early 1990's (see, e.g., Collins, J. E., etal., 1992, J. Vet. Diagn. Invest. 4:117-126); North American PRRS virusisolate MN-1b (Kwang, J. et al., 1994, J. Vet. Diagn. Invest.6:293-296); the Quebec LAF-exp91 strain of PRRS virus (Mardassi, H. etal., 1995, Arch. Virol. 140:1405-1418); and North American PRRS virusisolate VR 2385 (Meng, X.-J et al., 1994, J. Gen. Virol. 75:1795-1801).Additional examples of North American PRRS virus strains are describedherein. Genetic characteristics refer to genomic nucleotide sequencesimilarity and amino acid sequence similarity shared by North AmericanPRRS virus strains. Chinese PRRS virus strains generally evidence about80-93% nucleotide sequence similarity with North American strains.

“European PRRS virus” refers to any strain of PRRS virus having thegenetic characteristics associated with the PRRS virus that was firstisolated in Europe around 1991 (see, e.g., Wensvoort, G., et al., 1991,Vet. Q. 13:121-130). “European PRRS virus” is also sometimes referred toin the art as “Lelystad virus”. Further examples of European PRRS virusstrains are described herein.

A genetically modified virus is “attenuated” if it is less virulent thanits unmodified parental strain. A strain is “less virulent” if it showsa statistically significant decrease in one or more parametersdetermining disease severity. Such parameters may include level ofviremia, fever, severity of respiratory distress, severity ofreproductive symptoms, or number or severity of lung lesions, etc.

An “Infectious clone” is an isolated or cloned genome of the diseaseagent (e.g. viruses) that can be specifically and purposefully modifiedin the laboratory and then used to re-create the live geneticallymodified organism. A live genetically modified virus produced from theinfectious clone can be employed in a live viral vaccine. Alternatively,inactivated virus vaccines can be prepared by treating the live virusderived from the infectious clone with inactivating agents such asformalin or hydrophobic solvents, acids, etc., by irradiation withultraviolet light or X-rays, by heating, etc.

All currently available M.hyo vaccines are made from killed whole cellmycoplasma preparations (bacterins). In contrast, the present inventionemploys a soluble portion of a Mycoplasma hyopneumoniae (M.hyo) wholecell preparation, wherein the soluble portion of the M.hyo preparationis substantially free of both (i) IgG and (ii) immunocomplexes comprisedof antigen bound to immunoglobulin.

M.hyo has absolute requirements for exogenous sterols and fatty acids.These requirements generally necessitate growth of M.hyo inserum-containing media, such as porcine serum. Separation of theinsoluble material from the soluble portion of the M.hyo whole cellpreparation (e.g., by centrifugation, filtration, or precipitation) doesnot remove the porcine IgG or immune complexes. In one embodiment of thepresent invention, the M.hyo soluble portion is treated with protein-Aor protein-G in order to substantially remove the IgG and immunecomplexes contained in the culture supernatant. In this embodiment, itis understood that protein A treatment occurs post-M.hyo fermentation.This is alternatively referred to herein as downstream protein Atreatment. In another embodiment, upstream protein A treatment of thegrowth media (i.e., before M.hyo fermentation) can be employed. ProteinA binds to the Fc portion of IgG. Protein G binds preferentially to theFc portion of IgG, but can also bind to the Fab region. Methods forpurifying/removing total IgG from crude protein mixtures, such as tissueculture supernatant, serum and ascites fluid are known in the art.

In some embodiments, the soluble portion of the M.hyo preparationincludes at least one M.hyo protein antigen. In other embodiments, thesoluble portion of the M.hyo preparation includes two or more M.hyoprotein antigens.

In one embodiment, the M.hyo supernatant fraction includes one or moreof the following M.hyo specific protein antigens: M.hyo proteins ofapproximately 46 kD (p46), 64 kD (p64) and 97 kD (p9′7) molecularweights. In another embodiment, the supernatant fraction at leastincludes the p46, p64 and p97 M.hyo protein antigens. The M.hyo proteinof approximately 64 kD (p64) may be alternatively referred to herein asthe p65 surface antigen from M.hyo described by Kim et al. [Infect.Immun. 58(8):2637-2643 (1990)], as well as in U.S. Pat. No. 5,788,962.

Futo et al. described the cloning and characterization of a 46 kDsurface protein from M.hyo, which can be employed in the compositions ofthis invention [J. Bact 177: 1915-1917 (1995)]. In one embodiment, theM.hyo culture supernatant includes the p46 whose correspondingnucleotide and amino acid sequences from the P-5722 strain are set forthin SEQ ID NOs: 1 and 2, respectively. It is further contemplated thatvariants of such p46 sequences can be employed in the compositions ofthe present invention, as described below.

Zhang et al. described and characterized a p97 adhesin protein of M.hyo[Infect. Immun. 63: 1013-1019, 1995]. Additionally, King et al.described a 124 kD protein termed Mhp1 from the P-5722 strain of M.hyoand presented data suggesting that Mhp1 and p97 are the same protein[Vaccine 15:25-35 (1997)]. Such p97 proteins can be employed in thecompositions of this invention. In one embodiment, the M.hyo culturesupernatant includes the p97 whose corresponding nucleotide and aminoacid sequences from the P-5722 strain are set forth in SEQ ID NOs: 3 and4, respectively. It is further contemplated that variants of such p97sequences can be employed in the compositions of the present invention,as described below.

The M.hyo culture supernatant may include further M.hyo specific proteinantigens such as, but not limited to, proteins of approximately 41 kD(p41), 42 kD (p42), 89 kD (p89), and 65 kD (p65). See, Okada et al.,2000, J. Vet. Med. B 47:527-533 and Kim et al., 1990, Infect. Immun.58(8):2637-2643. In addition, the M.hyo culture supernatant can includeM.hyo specific protein antigens of approximately 102 kD (p102) and 216kD (p216). See, U.S. Pat. Nos. 6,162,435 and 7,419,806 to Minnion et al.

Any M.hyo strain may be used as a starting material to produce thesoluble portion of the M.hyo preparation of the present invention.Suitable strains of M.hyo may be obtained from commercial or academicsources, including depositories such as the American Type CultureCollection (ATCC) (Manassas, Va.) and the NRRL Culture Collection(Agricultural Research Service, U.S. Department of Agriculture, Peoria,Ill.). The ATCC alone lists the following six strains of M.hyo for sale:M.hyo ATCC 25095, M.hyo ATCC 25617, M.hyo ATCC 25934, M.hyo ATCC 27714,M.hyo ATCC 27715, and M.hyo ATCC 25934D. A preferred strain of M.hyo foruse in the embodiments of this invention is identified as strainP-5722-3, ATCC #55052, deposited on May 30, 1990 pursuant to theaccessibility rules required by the U.S. Patent and Trademark Office. Inview of the widespread dissemination of the disease, strains may also beobtained by recovering M.hyo from lung secretions or tissue from swineinfected with known strains causing mycoplasmal pneumonia in swine.

It is understood by those of skill in the art that variants of the M.hyosequences can be employed in the compositions of the present invention.Such variants could vary by as much as 10-20% in sequence identity andstill retain the antigenic characteristics that render it useful inimmunogenic compositions. Preferably, the M.hyo variants have at least80%, preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% sequence identify with the full-length genomicsequence of the wild-type M.hyo strain. The antigenic characteristics ofan immunological composition can be, for example, estimated by thechallenge experiment as provided in the Examples. Moreover, theantigenic characteristic of a modified M.hyo antigen is still retainedwhen the modified antigen confers at least 70%, preferably 80%, morepreferably 90% of the protective immunity as compared to the wild-typeM.hyo protein.

In one embodiment, M.hyo soluble p46 antigen is included in thecompositions of the invention at a final concentration of about 1.5μg/ml to about 10 μg/ml, preferably at about 2 μg/ml to about 6 μg/ml.It is noted that p46 is the protein used for the M.hyo potency test (seeexample section below). In another embodiment, the M.hyo antigen can beincluded in the compositions at a final amount of about 5.5% to about35% of the M.hyo whole culture protein A-treated supernatant.

The M.hyo soluble preparation of the present invention is both safe andefficacious against M.hyo and is suitable for single doseadministration. In addition, Applicants have surprisingly discoveredthat the M.hyo soluble preparation can be effectively combined withantigens from other pathogens without immunological interference betweenthe antigens. This makes the M.hyo soluble preparation of this inventionan effective platform for multivalent vaccines. The additional antigensmay be given concurrently with the M.hyo composition (i.e., as separatesingle vaccines) or combined in a ready-to-use vaccine.

In one embodiment, the immunogenic composition of the present inventionincludes at least one M.hyo soluble antigen and at least one additionalantigen. In one embodiment, the at least one additional antigen isprotective against a microorganism that can cause disease in pigs.

In some embodiments, the at least one additional antigen component isprotective against bacteria, viruses, or protozoans that are known toinfect pigs. Examples of such microorganisms include, but are notlimited to, the following: porcine circovirus type 2 (PCV2), porcinereproductive and respiratory syndrome virus (PRRSV), porcine parvovirus(PPV), Haemophilus parasuis, Pasteurella multocida, Streptococcum suis,Staphylococcus hyicus, Actinobacilllus pleuropneumoniae, Bordetellabronchiseptica, Salmonella choleraesuis, Salmonella enteritidis,Erysipelothrix rhusiopathiae, Mycoplama hyorhinis, Mycoplasmahyosynoviae, leptospira bacteria, Lawsonia intracellularis, swineinfluenza virus (SIV), Escherichia coli antigen, Brachyspirahyodysenteriae, porcine respiratory coronavirus, Porcine EpidemicDiarrhea (PED) virus, rotavirus, Torque teno virus (TTV), PorcineCytomegalovirus, Porcine enteroviruses, Encephalomyocarditis virus, apathogen causative of Aujesky's Disease, Classical Swine fever (CSF) anda pathogen causative of Swine Transmissable Gastroenteritis, orcombinations thereof

In one embodiment, an immunogenic composition of the present inventionincludes the combination of at least one M.hyo soluble antigen (e.g.,two or more) and a PCV2 antigen. In another embodiment, the compositionelicits a protective immune response in a pig against both M.hyo andPCV2.

In one embodiment, an M.hyo/PCV2 combination vaccine according to thepresent invention is provided as a single-dose, ready-to-use in onebottle vaccine. Such a ready-to-use combination vaccine requires nomixing of separate vaccines, so there is no risk of contamination oradditional labor associated with mixing and no requirement to use themixture within a few hours. Also, a one-bottle M.hyo/PCV2 combinationvaccine cuts waste and refrigerator storage space in half. Furthermore,one-dose administration eliminates the labor associated withadministering a second dose to the animal. It is noted that althoughPCV2/M.hyo combination vaccines currently exist, they are provided aseither a two-dose, ready-to-use vaccine (Circumvent® PCVM) or as asingle-dose, 2-bottle vaccine which requires the simultaneousadministration of separate vaccines (e.g., Ingelvac CircoFLEX® andIngelvac MycoFLEX®). Preferably, the M.hyo/PCV2 combination according tothe present invention would be compatible with other antigens, such asPRRS virus antigens, such that all antigens can be administered in asingle-dose.

In some embodiments, the PCV2 antigen component of an M.hyo/PCV2combination vaccine is in the form of a chimeric type-1-type 2circovirus. The chimeric virus includes an inactivated recombinantporcine circovirus type 1 expressing the porcine circovirus type 2 ORF2protein. Chimeric porcine circoviruses and methods for their preparationare described in WO 03/049703 A2, and also in U.S. Pat. Nos. 7,279,166and 7,575,752, which are incorporated herein by reference in theirentirety.

In one embodiment, the full-length DNA sequence of the genome of thechimeric PCV1-2 virus corresponds to SEQ ID NO: 5. or variants thereof,as described below. In another embodiment, the immunogenic ORF2 capsidgene of the chimeric PCV1-2 virus corresponds to SEQ ID NO: 6. In afurther embodiment, the amino acid sequence of the immunogenic ORF2protein expressed by the chimeric PCV1-2 virus corresponds to SEQ ID NO:7.

In yet another embodiment, the full-length DNA sequence of the genome ofthe chimeric PCV1-2 virus corresponds to SEQ ID NO: 8. In oneembodiment, the immunogenic ORF2 capsid gene of the chimeric PCV1-2virus corresponds to SEQ ID NO: 9. In a further embodiment, the aminoacid sequence of the immunogenic ORF2 protein expressed by the chimericPCV1-2 virus corresponds to SEQ ID NO: 10.

However, the PCV2 ORF2 DNA and protein of the chimeric PCV1-2 virus arenot limited to the sequences described above sincePCV2 ORF2 DNA andprotein is a highly conserved domain within PCV2 isolates.

In some embodiments, the PCV2 antigen component of an M.hyo/PCV2combination vaccine is in the form of a recombinant ORF2 protein. In oneembodiment, the recombinant ORF2 protein is expressed from a baculovirusvector. Alternatively, other known expression vectors can be used, suchas including, but not limited to, parapox vectors.

In one embodiment, the recombinant PCV2 ORF2 protein is that of SEQ IDNO: 11, which is encoded by SEQ ID NO: 12 (GenBank Accession No.AF086834). In another embodiment, the recombinant ORF2 protein is thatof SEQ ID NO: 13, which is encoded by SEQ ID NO: 14. In yet anotherembodiment, the recombinant ORF2 protein corresponds to SEQ ID NO: 15.In still another embodiment, the recombinant PCV2 ORF2 proteincorresponds to SEQ ID NO: 7. In a still further embodiment, therecombinant PCV2 ORF2 protein corresponds to SEQ ID NO: 10.

However, the present invention is not limited to the particular ORF2 DNAand protein sequences described above. Since PCV2 ORF2 DNA and proteinis a highly conserved domain within PCV2 isolates, any PCV2 ORF2 ishighly likely to be effective as the source of the PCV2 ORF2 DNA and/orpolypeptide as used in the chimeric PCV1-2 virus or in the recombinantPCV2 protein.

An example of a suitable PCV2 isolate from which the PCV2 ORF2 DNA andprotein sequences can be derived is PCV2 isolate number 40895 (depositedin the ATCC on Dec. 7, 2001 and assigned ATCC patent Deposit DesignationPTA-3914). The genomic (nucleotide) sequence of the PCV2 isolate number40895 is available under GenBank accession number AF264042. Otherexamples of suitable PCV2 isolates from which the PCV2 ORF2 DNA andprotein sequences can be derived include, but are not limited to, thefollowing: Imp.999, Imp.1010-Stoon, Imp.1011-48121, and Imp.1011-48285.The GenBank accession numbers of the genomic sequences corresponding tothese PCV2 isolates are AF055391, AF055392, AF055393 and AF055394,respectively.

In some forms, immunogenic portions of PCV2 ORF2 protein are used as theantigenic component in the composition. For example, truncated and/orsubstituted forms or fragments of PCV2 ORF2 protein may be employed inthe compositions of the present invention.

It is understood by those of skill in the art that variants of the PCV2sequences can be employed in the compositions of the present invention.Such variants could vary by as much as 10-20% in sequence identity andstill retain the antigenic characteristics that render it useful inimmunogenic compositions. Preferably, the PCV2 variants have at least80%, preferably at least 85%, more preferably at least 90%, even morepreferably at least 95% sequence identify with the full-length genomicsequence of the wild-type PCV2 isolate. The antigenic characteristics ofan immunological composition can be, for example, estimated by thechallenge experiment as provided in the Examples. Moreover, theantigenic characteristic of a modified PCV2 antigen is still retainedwhen the modified antigen confers at least 70%, preferably 80%, morepreferably 90% of the protective immunity as compared to the wild-typePCV2 ORF2 protein.

The PCV2 antigen component is provided in the immunogenic composition atan antigen inclusion level effective for inducing the desired immuneresponse, namely reducing the incidence of or lessening the severity ofclinical signs resulting from PCV2 infection.

In one embodiment, a chimeric PCV1-2 virus is included in thecompositions of the invention at a level of at least 1.0≦RP≦5.0, whereinRP is the Relative Potency unit determined by ELISA antigenquantification (in vitro potency test) compared to a reference vaccine.In another embodiment, a chimeric PCV1-2 virus is included in thecomposition of the invention at a final concentration of about 0.5% toabout 5% of 20-times (20×) concentrated bulk PCV1-2 antigen.

In another embodiment, the PCV2 ORF2 recombinant protein is included inthe compositions of the invention at a level of at least 0.2 μgantigen/ml of the final immunogenic composition (μg/ml). In a furtherembodiment, the PCV2 ORF2 recombinant protein inclusion level is fromabout 0.2 to about 400 μg/ml. In yet another embodiment, the PCV2 ORF2recombinant protein inclusion level is from about 0.3 to about 200μg/ml. In a still further embodiment, the PCV2 ORF2 recombinant proteininclusion level is from about 0.35 to about 100 μg/ml. In still anotherembodiment, the PCV2 ORF2 recombinant protein inclusion level is fromabout 0.4 to about 50 μg/ml.

In one embodiment, an immunogenic composition of the present inventionincludes the combination of at least one M.hyo soluble antigen (e.g.,two or more), a porcine circovirus type 2 (PCV2) antigen, and a PRRSvirus antigen. In another embodiment, the composition elicits aprotective immune response in a pig against M.hyo, PCV2 and PRRS virus.

In one embodiment, an M.hyo/PCV2/PRRS combination vaccine according tothe present invention is provided as a single-dose, 2-bottle vaccine.For example, in some embodiments, a M.hyo/PCV2 combination is providedas a stable liquid composition in a first bottle and a PRRS virus isprovided in a lyophilized state in a second bottle. In some embodiments,additional porcine antigens can be added to either the first or thesecond bottle.

In one embodiment, the PRRS virus component is provided as alyophilized, genetically modified live virus. Prior to administration,the M.hyo/PCV2 liquid from a first bottle can be used to re-hydrate thePRRS virus in a second bottle so that all three antigens can beadministered to the animal in a single-dose. It is noted that althoughPCV2/M.hyo/PRRS combination vaccines currently exist, they are providedas a single-dose, 3-bottle vaccine which requires the simultaneousadministration of three separate vaccines (e.g., Ingelvac CircoFLEX®,Ingelvac MycoFLEX® and Ingelvac®PRRS MLV).

The PRRS etiological agent was isolated for the first time in TheNetherlands, and named as Lelystad virus. This virus was described in WO92/21375 (Stichting Centraal Diegeneeskundig Instituut). An isolate ofthe European PRRS virus was deposited in the Institut Pasteur of Paris,number I-1102. The North American type was isolated almostsimultaneously with the isolation of the European type virus, and isdescribed in WO-93/03760 (Collins et al.) An isolate of the NorthAmerican type virus was deposited in the American Type CultureCollection (ATCC), number VR-2332.

Different strains have been isolated from both the European and NorthAmerican virus types. WO 93/07898 (Akzo) describes a European strain,and vaccines derived from it, deposited in CNCM (Institut Pasteur),number I-1140. Also, WO 93/14196 (Rhone-Merieux) describes a new strainisolated in France, deposited in CNCM (Institut Pasteur), number I-1153.Furthermore, EP0595436 B1 (Solvay) describes a new North American typestrain, more virulent than the one initially described, and vaccinesthereof. This strain has been deposited in ATCC, but the deposit numberis not detailed in the patent application. In addition, ES2074950 BA(Cyanamid Iberica) and its counterpart GB2282811 B2 describe a so-called“Spanish strain”, that is different from other European and NorthAmerican strains. This “Spanish strain” has been deposited in EuropeanAnimal Cell Culture Collection (EACCC), number V93070108.

Suitable PRRS virus antigens for use in the M.hyo/PCV2/PRRS compositionsof the present invention include North American PRRS virus isolates,Chinese PRRS virus strains, and European PRRS virus strains, as well asgenetically modified versions of such isolates/strains. In oneembodiment, the PRRS virus antigen component employed in thecompositions according to the present invention is a North American PRRSvirus.

In some embodiments, the PRRS virus antigen component employed in thecompositions of this invention is the North American PRRS virus isolatedesignated P129 or a live, genetically modified version thereof.Preferably, the genetically modified PRRS virus is unable to produce apathogenic infection yet is able to elicit an effective immunoprotectiveresponse against infection by the wild-type PRRS virus.

A genetically modified PRRS virus for use in the compositions of theinvention can be produced from an infectious clone. The preparation ofan infectious cDNA clone of the North American PRRS virus isolatedesignated P129 is described in U.S. Pat. No. 6,500,662 which is herebyincorporated fully by reference. The sequence of P129 cDNA is disclosedin Genbank Accession Number AF494042 and in U.S. Pat. No. 6,500,662.

In one embodiment, the nucleotide sequence of a non-virulent form ofP129 for use in the compositions of the present invention is representedby SEQ ID NO: 16. However, the present invention is not limited to thissequence. This sequence and the sequences of other non-virulent forms ofP129 are described in International Application No. PCT/IB2011/055003,filed Nov. 9, 2011, the contents of which (including any US NationalStage filings based on this International Application) are incorporatedherein by reference in their entirety. Preferably, the PRRS virus ismodified to prevent downregulation of interferon-mediated function.

In other embodiments, the PRRS virus antigen component employed in thecompositions of the invention is the PRRS virus isolate designatedISU-55. The ISU-55 isolate was deposited in the American Type CultureCollection (ATCC), under the accession number VR2430. The nucleotidesequence of the ORF2 to ORF5 genes of the ISU-55 isolate is representedby SEQ ID NO:17. The nucleotide sequence of the ORF6 and ORF7 genes ofthe ISU-55 isolate is represented by SEQ ID NO: 18.

Another suitable North American PRRS virus isolate which can be used inthe compositions is ISU-12, which was deposited in the ATCC under theaccession numbers VR2385 [3× plaque purified] and VR2386 [non-plaquepurified]. Still other suitable North American PRRS virus isolates whichcan be employed in the compositions of this invention are the following:ISU-51, ISU-3927, ISU-1894, ISU-22 and ISU-79, which were deposited inthe ATCC under the accession numbers VR2498, VR2431, VR2475, VR2429 andVR2474, respectively. Genetically modified versions of any of these ISUisolates can be employed in the compositions of this invention. TheseISU isolates and the ISU-55 isolate are described in detail in thefollowing U.S. patents to Paul, et al: U.S. Pat. Nos. 5,695,766,6,110,467, 6,251,397, 6,251,404, 6,380,376, 6,592,873, 6,773,908,6,977,078, 7,223,854, 7,264,802, 7,264,957, and 7,517,976, all of whichare incorporated herein by reference in their entirety.

In still other embodiments, the PRRS virus antigen component employed inthe compositions according to the present invention is the NorthAmerican type deposited in the American Type Culture Collection (ATCC),number VR-2332 or a genetically modified version thereof. For example,the PRRS virus can be a modified live virus based on the isolateidentified as ATCC VR2332, which is employed in INGELVAC® PRRS ATP andINGELVAC® PRRS MLV, from Boehringer Ingelheim Vetmedica, Inc.

In still other embodiments, the PRRS virus antigen component employed inthe compositions of the present invention is a European PRRS virusisolate or Lelystad virus or a genetically modified version thereof. Anexample of a suitable PRRS virus strain is identified as deposit No.I-1102, described above. Nucleotide and amino acid sequencescorresponding to the I-1102 deposit are described in U.S. Pat. No.5,620,691 to Wensvoort et al, which is hereby fully incorporated hereinby reference. The preparation of an infectious clone of a European PRRSvirus isolate or Lelystad virus is described in U.S. Pat. No. 6,268,199which is hereby fully incorporated herein by reference.

Other examples of suitable PRRS virus isolates include, but are notlimited to, those described above. Also, live, genetically modifiedversions of the PRRS virus isolates can be employed in the compositionsof the present invention. An infectious clone can be used to re-createsuch live genetically modified organisms.

It is understood by those of skill in the art that variants of the PRRSvirus sequences can be employed in the compositions of the presentinvention. Such variants could vary by as much as 10-20% in sequenceidentity and still retain the antigenic characteristics that render ituseful in immunogenic compositions. Preferably, the PRRS virus variantshave at least 80%, preferably at least 85%, more preferably at least90%, even more preferably at least 95% sequence identify with thefull-length genomic sequence of the wild-type PPRS virus isolate. Theantigenic characteristics of an immunological composition can be, forexample, estimated by challenge experiments. Moreover, the antigeniccharacteristic of a modified PRRS virus antigen is still retained whenthe modified antigen confers at least 70%, preferably 80%, morepreferably 90% of the protective immunity as compared to the wild-typePRRS virus antigen.

In one embodiment, the PRRS virus antigen component is a geneticallymodified, live virus which is included in the compositions of theinvention at a level of at least 2.1≦TCID₅₀≦5.2, wherein TCID₅₀ is thetissue culture infectious dose 50% determined by antigen quantification(in vitro potency test).

The PCV2 antigen component of the M.hyo/PCV2/PRRS compositions of theinvention can be in the form of a chimeric type-1-type 2 circovirus, thechimeric virus including an inactivated recombinant porcine circovirustype 1 expressing the porcine circovirus type 2 ORF2 protein. In anotherembodiment, the PCV2 antigen component of the M.hyo/PCV2/PRRScompositions of the invention is in the form of a recombinant ORF2protein.

Suitable PCV2 antigens for use in the M.hyo/PCV2/PRRS compositions canbe derived from any of the PCV2 isolates described above, as well asother PCV2 isolates. Suitable PCV2 antigens to be employed in thecompositions of the invention include, but are not limited to, the PCV2sequences described above and variants thereof.

Vaccines of the present invention can be formulated following acceptedconvention to include acceptable carriers for animals, including humans(if applicable), such as standard buffers, stabilizers, diluents,preservatives, and/or solubilizers, and can also be formulated tofacilitate sustained release. Diluents include water, saline, dextrose,ethanol, glycerol, and the like. Additives for isotonicity includesodium chloride, dextrose, mannitol, sorbitol, and lactose, amongothers. Stabilizers include albumin, among others. Other suitablevaccine vehicles and additives, including those that are particularlyuseful in formulating modified live vaccines, are known or will beapparent to those skilled in the art. See, e.g., Remington'sPharmaceutical Science, 18th ed., 1990, Mack Publishing, which isincorporated herein by reference.

Vaccines of the present invention can further comprise one or moreadditional immunomodulatory components such as, e.g., an adjuvant orcytokine, among others. Types of suitable adjuvants for use in thecompositions of the present invention include the following: anoil-in-water adjuvant, a polymer and water adjuvant, a water-in-oiladjuvant, an aluminum hydroxide adjuvant, a vitamin E adjuvant andcombinations thereof. Some specific examples of adjuvants include, butare not limited to, complete Freund's adjuvant, incomplete Freund'sadjuvant, Corynebacterium parvum, Bacillus Calmette Guerin, aluminumhydroxide gel, glucan, dextran sulfate, iron oxide, sodium alginate,Bacto-Adjuvant, certain synthetic polymers such as poly amino acids andco-polymers of amino acids, Block copolymer (CytRx, Atlanta, Ga.), QS-21(Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, EmeryvilleCalif.), AMPHIGEN® adjuvant, saponin, Quil A or other saponin fraction,monophosphoryl lipid A, and Avridine lipid-amine adjuvant(N,N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), “REGRESSIN”(Vetrepharm, Athens, Ga.), paraffin oil, RIBI adjuvant system (RibiInc., Hamilton, Mont.), muramyl dipeptide and the like.

Non-limiting examples of oil-in-water emulsions useful in the vaccine ofthe invention include modified SEAM62 and SEAM ½ formulations. ModifiedSEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma),1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM ½ is anoil-in-water emulsion comprising 5% (v/v) squalene, 1% (v/v) SPAN® 85detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 μg/mlQuil A, and 50 μg/ml cholesterol.

Another example of an adjuvant useful in the compositions of theinvention is SP-oil. As used in the specification and claims, the term“SP oil” designates an oil emulsion comprising apolyoxyethylene-polyoxypropylene block copolymer, squalane,polyoxyethylene sorbitan monooleate and a buffered salt solution.Polyoxyethylene-polyoxypropylene block copolymers are surfactants thataid in suspending solid and liquid components. These surfactants arecommercially available as polymers under the trade name Pluronic®. Thepreferred surfactant is poloxamer 401 which is commercially availableunder the trade name Pluronic® L-121. In general, the SP oil emulsion isan immunostimulating adjuvant mixture which will comprise about 1 to 3%vol/vol of block copolymer, about 2 to 6% vol/vol of squalane, moreparticularly about 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol ofpolyoxyethylene sorbitan monooleate, with the remainder being a bufferedsalt solution. In one embodiment, the SP-oil emulsion is present in thefinal composition in v/v amounts of about 1% to 25%, preferably about 2%to 15%, more preferably about 5% to 12% v/v.

Yet another example of a suitable adjuvant for use in the compositionsof the invention is AMPHIGEN™ adjuvant which consists of de-oiledlecithin dissolved in an oil, usually light liquid paraffin.

Other examples of adjuvants useful in the compositions of the inventionare the following proprietary adjuvants: Microsol Diluvac Forte® duelemulsion adjuvant system, Emunade adjuvant, and Xsolve adjuvant. Boththe Emunade and Xsolve adjuvants are emulsions of light mineral oil inwater, but Emunade also contains alhydrogel, and d,l-α-tocopherylacetate is part of the XSolve adjuvant. A still further example of asuitable adjuvant for use in the compositions of the invention isImpranFLEX™ adjuvant (a water-in-oil adjuvant). A still further exampleof a suitable adjuvant is a Carbomer (Carbopol®) based adjuvant.Preferred Carbopol® adjuvants include Carbopol® 934 polymer andCarbopol® 941 polymer.

In one embodiment, the adjuvant or adjuvant mixture is added in anamount of about 100 μg to about 10 mg per dose. In another embodiment,the adjuvant/adjuvant mixture is added in an amount of about 200 μg toabout 5 mg per dose. In yet another embodiment, the adjuvant/adjuvantmixture is added in an amount of about 300 μg to about 1 mg/dose.

The adjuvant or adjuvant mixture is typically present in the vaccinecomposition of the invention in v/v amounts of about 1% to 25%,preferably about 2% to 15%, more preferably about 5% to 12% v/v.

Other “immunomodulators” that can be included in the vaccine include,e.g., one or more interleukins, interferons, or other known cytokines.In one embodiment, the adjuvant may be a cyclodextrin derivative or apolyanionic polymer, such as those described in U.S. Pat. Nos. 6,165,995and 6,610,310, respectively.

A further aspect relates to a method for preparing an immunogeniccomposition according to the present invention. This method comprises i)culturing M.hyo in a suitable media over periods ranging from 18-144hours; ii) subsequently inactivating the M. hyo culture; iii) harvestingthe inactivated culture fluid, wherein the inactivated culture fluidcomprises an M.hyo whole cell preparation comprising both a solubleliquid fraction and insoluble cellular material; iv) separating thesoluble liquid fraction from the insoluble cellular material; and v)substantially removing both IgG and antigen/immunoglobulinimmunocomplexes from the separated soluble liquid fraction.

An example of a suitable media for culturing M.hyo is PPLO Broth(Mycoplasma Broth Base), which when supplemented with nutritiveenrichments, is used for isolating and cultivating Mycoplasma.

In some embodiments, the culture of M.hyo is grown until late log phasegrowth, after which the culture is inactivated. In some otherembodiments, the culture is inactivated by raising the pH (e.g., toabout 7.8). This occurs by exposing the production culture to aninactivation agent, such as binary ethylenimine (BEI). The BEI isgenerated in situ during incubation of L-bromoethylamine hydrobromide(BEA) in the production culture. Subsequently, the pH of the inactivatedculture is neutralized, such as by adding an equivalent amount of anagent that neutralizes the inactivation agent within the solution. Insome embodiments, the inactivation agent is BEI and the neutralizationagent is sodium thiosulfate. In one embodiment, the pH of theinactivated culture is adjusted to about 7.4 by adding sodiumthiosulfate.

In some embodiments, the soluble liquid fraction of the M.hyo whole cellpreparation is separated from the insoluble cellular material usingconventional methods. In one embodiment, this separation is by afiltration step. In another embodiment, this separation is by acentrifugation step. In yet another embodiment, the separation is by aprecipitation step.

In one embodiment, the soluble liquid fraction of an inactivated,neutralized M.hyo whole cell preparation is treated with Protein A resinto substantially remove both the IgG and antigen/immunoglobulinimmunocomplexes therein. In other embodiments, Protein G resin can beused to substantially remove both the IgG and antigen/immunoglobulinimmunocomplexes contained in the soluble liquid fraction. Methods forremoving both IgG and antigen/immunoglobulin immunocomplexes with eitherProtein A or Protein G resins are well known in the art.

According to a further aspect, the method for preparing an immunogeniccomposition, such as a vaccine, according to the invention comprisespreparing the soluble M.hyo antigen as described above and mixing thiswith a suitable adjuvant and one or more pharmaceutically-acceptablecarriers. This method optionally includes adding at least one additionalporcine antigen, such as a PCV2 antigen and/or PRRS virus antigen asdescribed above.

A further aspect of the present invention relates to a kit. A “kit”refers to a plurality of components which are grouped together. In oneembodiment, a kit according to the present invention includes a bottle(or other suitable receptable) comprising an immunogenic compositionincluding the soluble portion of a Mycoplasma hyopneumoniae (M.hyo)whole cell preparation, wherein the soluble portion of the M.hyopreparation is substantially free of both (i) IgG and (ii)antigen/immunoglobulin immunocomplexes. Optionally, the kit can furtherinclude an instruction manual. The instruction manual includes theinformation to administer the immunogenic composition.

In some embodiments, the bottle containing the soluble portion of theM.hyo preparation further includes PCV2 antigen. In some embodiments,the M.hyo/PCV2 combination in the bottle is provided as a ready-to-useliquid composition.

In other embodiments, the kit includes a second bottle comprising PRRSvirus antigen. In some embodiments, the PRRS virus antigen is in theform of a genetically modified, live virus which is provided in alyophilized state. In such instances, the instruction manual willinclude the directions for re-hydrating the PRRS virus component withthe liquid contents from a bottle containing the M.hyo/PCV2 combination.The instruction manual will also include the information to administerthe resultant M.hyo/PCV2/PRRS trivalent formulation(s).

In some embodiments, an immunogenic composition according to thisinvention is administered to pigs having maternally derived antibodiesagainst M.hyo. In other embodiments, an immunogenic composition of thepresent invention is administered to pigs having maternally derivedantibodies against both M.hyo and at least one other microorganism thatcan cause disease in pigs.

In some embodiments, an immunogenic composition of the presentinvention, such as a monovalent or multivalent vaccine, is administeredto a piglet aged 3 weeks or older. However, it is contemplated that amonovalent or multivalent vaccine composition according to the inventionmay also be used to re-vaccinate gilts pre-breeding. As is known in theart, a gilt is a female pig that has never been pregnant. Vaccinatedgilts will pass maternally derived antibodies onto their sucklingnewborns via colostrum.

It is further contemplated that a monovalent or multivalent vaccineaccording to the invention can be used to annually re-vaccinate breedingherds. Preferably, a monovalent or multivalent vaccine according to thepresent invention is administered to pigs (e.g., piglets or gilts) inone dose. In one embodiment, a multivalent vaccine according to thepresent invention does not require mixing of separate monovalentvaccines prior to administration, i.e., it is provided as a ready-to-useformulation. In another embodiment, a multivalent formulation requiresmixing of a divalent vaccine contained in a first bottle with amonovalent vaccine contained in a second bottle. Optionally, additionalantigens can be added to either of these bottles.

In some embodiments, the onset of immunity is from 2-3 weekspost-vaccination with a monovalent or multivalent vaccine compositionaccording to the present invention. In other embodiments, the durationof immunity is about 17-23 weeks post-vaccination with a monovalent ormultivalent vaccine composition according to the present invention.

The following examples set forth preferred materials and procedures inaccordance with the present invention. However, it is to be understoodthat these examples are provided by way of illustration only, andnothing therein should be deemed a limitation upon the overall scope ofthe invention.

EXAMPLES Example 1 Mycoplasma hyopneumoniae Production Methods for PCV2Combinable M.hyo Antigen

M.hyo Fermentation and Inactivation

Media for seed scale and antigen production was prepared as follows.Porcine heart derived Pleuropenumonia-like Organism (PPLO) Broth (BDBiosciences catalog No. 21498) was made per manufacturer's directions(i.e., 21 g/L) and yeast extract solution was made at 21 g/L in USP.Yeast extract solution was then added to the PPLO at 6.25% and themixture was sterilized by heating to 121° C. for ≧30 minutes. Cysteinehydrochloride was prepared at 90 g/L and filter sterilized. Dextrosesolution was made by adding 450 g of dextrose per liter of USP waterfollowed by heat sterilization. To prepare the final medium, porcineserum was added to the base medium at 10% followed by cysteine at 0.01%and dextrose at 1.0%. The medium was inoculated with a 10% v:v of a logphase culture of M. hyopeumoniae (strain P-5722-3). The culture was heldat 37° C. and pH and dO were maintained at 7.0 and 25%, respectively. Atlate log phase growth, the culture was inactivated was inactivated bybinary ethylenimine (BEI), an aziridine compound, produced from2-bromoethylamine hydrobromide. Specifically, the inactivation occurredby raising the pH to 7.8 by adding 2-bromoethylaminehydrobromide (BEA)to a final concentration of 4 mM and incubating for 24 hours. The BEIwas neutralized by addition of sodium thiosulfate at a 1:1 molar ratiofollowed by additional 24 hour incubation. The inactivated culture fluidwas held at 2-8° C. until further processing.

Example 2 Chimeric Porcine Circovirus (cPCV) 1-2 Production Methods

The cPCV1-2 was constructed by cloning the immunogenic capsid gene ofthe pathogenic porcine circovirus type 2 (PCV2) into the genomicbackbone of the nonpathogenic porcine circovirus type 1 (PCV1). Theprocedure for construction of the chimeric DNA clone is described, forexample, in U.S. Pat. No. 7,279,166, which is incorporated herein byreference in its entirety. An infectious stock of the chimeric virus wasacquired from Dr. X. J. Meng, Virginia Polytechnic Institute and StateUniversity, Blacksburg, Va., and was used to infect Porcine Kidney(PK)-15 cells grown in Minimum Essential Medium (MEM) supplemented with0.05% lactalbumin hydrolysate (LAH), 30 μg/mL gentamicin sulfate, and 5%fetal bovine serum. The resulting cPCV1-2 infected PK-15 cells werefurther expanded by serial passing four more times using the same growthmedium except with 2-3% fetal bovine serum. The fifth passage wasfrozen, thawed and filtered, and the resulting lysates were used toprepare a pre-master seed and subsequent master seed.

The medium which was used for producing virus seeds was the same as thatused in producing virus stock. For the growth medium, MEM, OptiMEM, orequivalent is the basal medium which can be used for planting the PK-15cell line for outgrowth. The growth medium can be supplemented with upto 10% bovine serum, up to 0.5% lactalbumin hydrolysate, up to 0.5%bovine serum albumin, and up to 30 μg/mL gentamicin. For the viruspropagation medium, MEM, OptiMEM, or equivalent is used. The viruspropagation medium can be supplemented with up to 0.5% lactalbuminhydrolysate, up to 2% bovine serum, up to 0.5% bovine serum albumin, andup to 30 μg/mL gentamicin. Up to 5 g/L glucose and up to 5 mmol/LL-glutamine can be added to the growth medium and/or the viruspropagation medium as required to sustain the cells.

The cPCV1-2 master seed virus are added to a cell suspension of PK-15cells and adsorbed for up to 3 hours. Seed virus is diluted in growthbasal medium to provide a multiplicity of infection (MOI) of 0.1-0.0001.

Cultures of PK-15 cells are initially inoculated with working seed virusat the time of cell planting, or when cells reach approximately 20% to50% confluency. This initial passage may be referred as “One-StepInfection Method” for the production of antigen stock, or may be furtherused for serial passages. For serial passages, the cPCV1-2 infectedPK-15 cells are further expanded up to passage 7 by serial splits at theratio of 1:5-20 for virus propagation. Culture medium containing aninfected cell suspension from the previous passage serves as seedmaterial for the next passage. The cPCV1-2 infected cells are incubatedfor three (3) to 14 days for each passage at 36±2° C. when cells reach≧90% confluency. The cPCV1-2 virus causes observable cytopathic changesduring viral replication. At harvest, rounding of cells and considerablefloating debris is observed. Cultures are also observed for visualevidence of bacterial or fungal contamination. The incubation timebetween harvests for the cPCV antigen is provided in Table 1 below:

TABLE 1 Minimum and Maximum Times for Harvesting cPCV Antigen Minimum/Maximum Temperature Method Time Range One-Step Infection  5 to 16 days36 ± 2° C. Serial Passage (MSV + 3 to 16 to 36 Days 36 ± 2° C. MSV + 7)

The cPCV1-2 culture fluids are harvested into sterile vessels and aresampled for mycoplasma testing using known methods. Multiple harvestsmay be conducted from roller bottles, bioreactors and perfusion vessels.

Prior to inactivation of the harvested cPCV1-2 virus, one or moreantigen lots may be concentrated (e.g., up to 60×) by ultrafiltration.The concentrates may be washed with balanced salt solution to reduceserum proteins.

The method of inactivation, attenuation, or detoxification of thecPCV1-2 virus will now be described. After cPCV antigen concentration,Beta-propiolactone (BPL) is added to the pooled cPCV1-2 viral materialto obtain an approximate concentration of 0.2% v/v. The pooled viralfluids are then agitated for a minimum of 15 minutes and then theinactivating bulk antigen fluids are transferred to a second sterilevessel. The transferred antigen fluids are maintained at 2-7° C., withconstant agitation, for a minimum of 24 hours. After a minimum of 24hours, a second addition of 0.2% v/v of BPL is added to the pooledsuspension. The contents are subsequently agitated, transferred to athird vessel, and maintained at 2-7° C., with constant agitation, for anadditional time of not less than 84 hours. In general, the totalinactivation time is not less than 108 hours and not more than 120hours. The inactivation method is summarized in Table 2 below.

TABLE 2 Inactivation Method Time- Final Hours Inactivant ConcentrationTemp. Range (Min/Max) Beta- 0.4% v/v 2-7° C. 108-120 propiolactone (2 ×0.2% v/v (w/Agitation) (BPL) additions)

The inactivation is terminated by the addition of a final concentrationof not more than 0.1 M solution of sodium thiosulfate. The pH of theinactivated antigen stock is adjusted to about 6.8 using NaOH or HCl.Following inactivation, a representative sample is taken from the pooland tested for completion of inactivation. The inactivated cPCV 1-2antigen product is standardized to a meet a target of greater than 1.0RP as measured via potency ELISA.

Example 3 Down Stream Processing of M. Hyo Antigens and AnalyticalTesting of these Processed Antigens

Down Stream Processing of M.hyo Antigens:

Inactivated fermentation fluid (prepared as described above inExample 1) was treated for each indicated group as follows. Theseprocessed M.hyo antigens were employed in Example 4 below.

-   T02: (Whole Bulk) Not processed.-   T03: (10×UF concentrated) Concentrated via tangential flow    filtration via a 100 KDa molecular weight cutoff membrane (hollow    fiber). Final volume reduction was equal to 10×.-   T04 & T05: (10×UF concentrated & centrifuged) Concentrated    mycoplasma cells (from T03) were collected and washed one time with    PBS via centrifugation at ˜20,000×g (Sorvall model RC5B).-   T06 & T07: (10× centrifuged) Inactivated fermentation fluid was    centrifuged at ˜20,000×g (Sorvall RC5B) and washed one time by    resuspending the cells in PBS followed by an additional    centrifugation. Final volume reduction was equal to 10×.-   T08: (10× centrifuged & Heated) Mycoplasma cells were concentrated    and washed per T06 and heated to 65° C. for 10 minutes.-   T09: (Cell-free supernatant) Supernatant collected from the first    centrifugation as described for T06 was filter sterilized through a    0.2 micron filter (Nalgene).-   T10: (Cell-free supernatant-Protein-A treated) Sterile supernatant    (prepared per T09) was mixed with Protein A resin (Protein A    Sepharose, Pharmacia Inc) at a 10:1 volume ratio for 4 hours. Resin    was removed sterile filtration and filtered fluid was stored at    2-8° C. This process uses post-fermentation “downstream” protein A    treatment to remove antibodies and immunocomplexes. Although the    present invention does not preclude upstream protein A treatment,    the present inventors have found that in the case of M.hyo, upstream    protein A treatment of the growth media led to p46 results which    were lower and inconsistent as compared to untreated media (data not    shown).    Analytical Testing of M.hyo Downstream Processed Antigens

The downstream processed M.hyo antigens preparations (prepared asdescribed above) were tested for the recovery of M.hyo specific p46antigen, and the presence of PCV2 antibody. In addition, these M.hyoantigen preparations were tested for the presence of Torque Teno Virus(TTV), including genotype 1 (g1TTV) and genotype 2 (g2TTV). The resultsare presented below in Table 3.

TABLE 3 Characterization of M. hyo Downstream Processed Antigens Bulk M.Hyo PCV2 ab qPCR DNA Treatment p46 RU/mL SIP ratio g1TTV g2TTV Wholebulk 809 0.248 1.00E+03 1.78E+03 10x UF 6666 0.819 1.00E+03 9.94E+03concentrated 10x UF conc. + 614 0.019 0 0 Centrifuge 10x 763 −0.0151.90E+02 1.91E+02 Centrifuged 10x 690 −0.012 0 2.07E+02 Centrifuged +Heated Cell-free supe 719 0.242 4.20E+02 3.23E+03 Cell-free supe 826−0.014 0 2.06E+03 (Prot A)

With reference to Table 3 above, recovery of the M.hyo-specific p46antigen was demonstrated for each of the M.hyo downstream processedantigen preparations. In addition, the following treatments successfullyremoved PCV2 antibody: 10×UF concentrated & centrifuged, 10×centrifuged, 10× centrifuged & heated and Cell-free supernatant(Protein-A treated). With respect to TTV, the following treatmentssuccessfully removed g1TTV: 10× UF concentrated & centrifuged, 10×centrifuged & heated, and Cell-free supernatant (Protein-A treated).Only the treatment designated 10× UF concentrated & centrifuged removedg2TTV. Torque teno virus isolates, including genotypes 1 and 2 aredescribed in US20110150913, which is incorporated herein by reference inits entirety.

Since it is known in the art that Protein A binds IgG, it is understoodby those of ordinary skill in the art that not only PCV2 antibody, butother swine antibodies, including PRRS antibody, HPS antibody, and SIVantibody will be effectively removed by the Protein-A treatment. Thismakes the Cell-free Protein-A treated M.hyo supernatant of thisinvention compatible not only with PCV2 antigen, but also with otherporcine antigens due to the lack of immunological interference betweenthe antigens. Additionally, the removal of the non-protective celldebris and removal of the immunoglobulin and antigen/immunoglobulincomplexes is reasonably expected to make a safer vaccine.

Example 4 Preparation of M.hyo Experimental Vaccine Formulations

All experimental M.hyo vaccines were formulated with a finalconcentration of 5% Amphigen adjuvant. In addition, all vaccines werestandardized with a p46 ELISA and preserved with thimerosol. Theexperimental vaccine formulations were prepared with M.hyo antigensprocessed according to treatments T02-T10 above. In addition, TreatmentT01 corresponded to a placebo (no

M.hyo antigen, only 5% Amphigen adjuvant) whereas Treatment T11 is apositive control corresponding to an expired bacterin-based M.hyovaccine (RespiSure-ONE®, Pfizer Animal Health). These formulations aredescribed in Table 4 below.

TABLE 4 M. hyo Experimental Vaccine Formulations M Hyo Adju- Formula-Target p46 antigen vant tion Treatment IVP Serial* units/ds (mL) (mL)Vol. (mL) T01 123639 (Placebo) 5% Amphigen only, No Antigen T02 L100211A452 279.36 250 1000 T03 L100211B 452 6.78 50 200 T04 L100211C 452 73.6250 200 T05 L100211D 816 132.90 50 200 T06 L100211E 452 59.24 50 200 T07L100211F 816 106.95 50 200 T08 L100211G 452 65.51 50 200 T09 L100211H452 62.87 50 200 T10 L100211J 452 54.72 50 200 T11 A827870 Expired“RespiSure” vaccine *Investigational Veterinary Product (IVP) Serial

Example 5 Evaluation of the In Vivo Efficacy of M.hyo Vaccines withM.hyo Antigens from Different Downstream Processes

This study was conducted to evaluate the in vivo efficacy of Mycoplasmahyopneumoniae (M hyo) vaccines with M hyo antigens from differentdownstream processes (DSP). Pigs at 3 weeks of age were intramuscularlyinoculated with a single dose of the different vaccine formulationsdescribed in Table 4 above. Sixteen animals were included in each of thetreatment groups. Animals were challenged 21 days after vaccination witha virulent M.hyo field isolate. Animals were necropsied 28 days afterchallenge and the lungs were removed and scored for consolidationconsistent with M.hyo infection. The primary criterion for protectionagainst M.hyo challenge was lung consolidation scores. It is generallyaccepted that there is a relationship between the size of the lunglesions caused by enzootic pneumonia and an adverse effect on growthrate. Table 5 below contains the lung lesion scores for the respectivetreatment groups. Statistical significance was determined by a MixedModel Analysis of lung scores for each group.

TABLE 5 Lung Lesion Results % Lung p46 RP Lesions Back Range % Target/Transformed Lung with Treatment Description Observed LS Means LesionsContrast p-value Significant T01 Placebo (5% N/A 11.7 1.2- 4.3 N/A N/AN/A Amphigen) T02 Whole bulk 13/15.6 1.2  0.1-18.5 T01 vs 02  0 Yes T03Whole bulk 13/11.9 0.3 0.0-2.8 T01 vs 03  0 Yes UF 10x T04 UF 10x +13/28.1 5.9 0.0-40.5 T01 vs 04  0.1589 No Centrifuged T05 UF 10x +24/48.2 3.7 0.0-42.3 T01 vs T05 0.0309 Yes Centrifuged T06 10xCentrifuged 13/30.4 4.7 0.0-23.6 T01 vs 06  0.0388 Yes T07 10xCentrifuged 24/57.4 4.6 0.3-37.3 T01 vs T07 0.0323 Yes T08 10xCentrifuged + 13/17.7 4.5 0.3-21.7 T01 vs T08 0.0137 Yes Heat T09Supernatant 13/14.1 1.4 0.0-33.0 T01 vs T09 0.0004 Yes (no cells) T10Supernatant + 13/12.1 3.1 0.0-25.8 T01 vs T10 0.0094 Yes Prot A T11Expired RSO 13/12.5 2.2 0.1-32.1 T01 vs T11 0.0009 Yes

With reference to Table 5 above, the results with M.hyo antigens fromdifferent downstream processes indicated that all experimental vaccinesexcept T04 significantly differed from the placebo. These M.hyo lesionresults are depicted graphically in FIG. 1. As shown in FIG. 1, T04 gaveunacceptable results. All other treatments differed significantly fromthe placebo (T01). The lung consolidation scores indicated that T02, T03and T09-T11 gave the most efficacious protection against M.hyochallenge.

The p46 relative potency of the experimental vaccines was assessed byusing a double antibody sandwich enzyme-linked immunosorbent assay (DASELISA). The p46 DAS ELISA results presented in Table 5 above indicatethat all the experimental vaccines exceeded the target potency. Inaddition, the p46 relative potency was either maintained or increasedduring storage of the vaccines over a one-month period (data not shown).A perceived increase in potency over time was observed in centrifugedantigens with the exception of those antigens that were subjected toheat. While not wishing to be bound by any one theory, it is likely thatcell “carcasses” are breaking up over time and released more of themembrane bound p46 antigen in the case of the centrifuged antigens.

Example 6 Evaluation of the Compatibility of the Experimental M.hyoVaccines with PCV2 Antigen

This study was conducted to evaluate the compatibility of the M.hyoexperimental vaccines with M hyo antigens from different downstreamprocesses with PCV2 antigen. The M.hyo experimental vaccine formulationsare described in Tables 4 and 5 above. The observed p46 relativepotencies for these vaccines are described in Table 5 above. These M.hyoexperimental vaccines were each combined with PCV2 antigen. In thisexample, the PCV2 antigen was a killed PCV Type 1-Type 2 chimeric virus(Fostera PCV) prepared as described above in Example 2. The chimericvirus was included in the compositions at an initial level of about1.6≦RP, wherein the RP is the Relative Potency unit determined by PCV2ELISA antigen quantification (in vitro potency test) compared to anefficacious reference vaccine.

The experimental M.hyo/PCV2 combination formulations were evaluated byPCV2 ELISA. The results are presented in FIG. 2. As shown in FIG. 2,only the M.hyo antigen preparations from the following downstreamprocesses were compatible with the PCV2 antigen: Ultrafiltration &Centrifugation (T04 & T05), Centrifugation (T06 & T07), Centrifugationplus heat (T08) and Protein A-treated Supernatant (T10). Of these, theM.hyo Protein A-treated supernatant was the most compatible with PCV2antigen when compared to the placebo control which included the chimericvirus and Amphigen adjuvant, but no M.hyo antigen. The level of chimericPCV virus in the Protein-A treated supernatant was 1.5 RP as compared to1.69 RP for the placebo. It was therefore concluded that there is no orminimal immunological interference between the Protein-A treated M.hyosoluble antigen preparation and PCV2 antigen of the chimeric virus.

The in vivo efficacy of the Protein-A treated M.hyo supernatantdemonstrated in Example 5 above together with the results described inthe present example indicated that the Protein-A treated supernatant wasa potentially effective platform for M.hyo-PCV2 combinations.

Example 7 Evaluation of PCV2 Efficacy of a 1-Bottle PCV2/M.hyoCombination Vaccine in Different Adjuvant Formulations

This study was designed to evaluate the PCV2 efficacy in a 1-bottlePCV2/M.hyo combination vaccine in different adjuvant formulations. Inthis example, the PCV2 antigen was a killed PCV Type 1-Type 2 chimericvirus (Fostera PCV). The chimeric virus was combined with an M.hyosoluble antigen preparation that was substantially free of IgG (i.e.,Protein A-treated supernatant).

Processing of Fluids:

Inactivated M.hyo fermentation fluid (described above in Example 1) wastreated for each indicated group as follows.

-   T02-T04: Whole fermentation fluid containing live M. hyopneumoniae    cells (described above) was centrifuged at ˜20,000×g (Sorvall RCSB)    and the supernatant collected and sterilized through a 0.2 μM    filter. rProtein A Sepharose (part number 17-5199-03, GE Healthcare)    was packed into a 1 L chromatography column. After removal of the    storage buffer and treatment with 2 column volumes of 1M acetic    acid, the resin was equilibrated with 5 column volumes of 50 mM    NaPO4/1M NaCl buffer, pH 7.04. Approximately 2 liters of the    clarified/filtered M. hyopneumoniae antigen containing fluids were    passed through the Protein A resin at a flow rate of 100 cm/hr. The    flow through was collected and sterilized via 0.2 μM filter.-   T05: This is a positive control corresponding to a Fostera PCV-like    formulation (no M.hyo antigen). The level of the chimeric virus in    this Fostera PCV-like formulation was approximately at Minimum    Immunizing Dose (MID) formulation levels. The chimeric virus was    included in the PCV2/M.hyo experimental vaccines at similar    formulation levels.

All experimental PCV2/M.hyo vaccines were formulated with differentadjuvant formulations. The experimental vaccine formulations wereprepared with M.hyo antigens processed according to treatments T02-T04above. In addition, Treatment T01 corresponded to a placebo (sterilesaline).

All vaccines were standardized with a p46 ELISA and preserved withthimerosol.

These experimental formulations are described in Table 6 below, whereinthe symbol * indicates the M hyo antigen from global M hyo seed, ProteinA treated supernatant and the symbol ** indicates InvestigationalVeterinary Product (IVP) serial.

TABLE 6 PCV2/M. hyo Experimental Vaccine Formulations Used for PCV2Efficacy Study IVP PCV1-2 M Hyo* Adju- Treatment Serial** Ag Ag vantOther T01 87-244-DK NA Sterile (Placebo) Saline T02 L0411RK08 1.6 RP 7.5RP 10% SP Oil NA T03 L0411RK09 5% Amphigen T04 L0611RK03 5% Amphigen +5% SLCD T05 L0611RK04 NA 20% SLCD

Pigs at 3 weeks of age were intramuscularly inoculated with a singledose of the different vaccine formulations described in Table 6 above.Sixteen animals were included in each of the treatment groups. Animalswere challenged 21 days after vaccination with a virulent PCV2 fieldisolate.

FIG. 3 is a graph showing the PCV2 viremia results (PCV2 QuantitativePCR) observed with the different adjuvant platforms. It is noted thatPCV2 viremia was used as the primary efficacy variable. The PCV2 viremiaresults are presented as DNA copies/ml. As shown in FIG. 3, alltreatments had significantly less viremia compared to the placebo ondays 28, 35 and 42 (challenge was day 21). The 10% SP-oil adjuvant hadsignificantly less viremia compared to 5% Amphigen at Days 28 and 35.The 5% Amphigen plus 5% SLCD adjuvant had significantly less viremiacompared to 5% Amphigen at Days 28 and 35. The 20% SLCD adjuvantplatform had significantly less viremia compared to 5% Amphigen at Days28, 35 and 42.

PCV2 Serology, PCV2 fecal shed, PCV2 nasal shed, Cell Mediated Immune(CMI) responses, lymphoid depletion, and Immunohistochemistry (IHC) werealso monitored as secondary efficacy variables. These results will nowbe described below.

FIG. 4 is a graph showing the PCV2 ELISA results on days 1, 20 and 42 ofthe study (challenge was day 21). The status of each sample wasexpressed as a sample to positive ratio (S/P). As shown in FIG. 4, 20%SLCD was the only treatment which was significantly different from theplacebo (T01) at both day 20 and day 42. Also, 5% Amphigen was the onlytreatment not significantly different from the placebo at day 20.

FIG. 5 is a graph showing the PCV2 fecal shed obtained with the T02-T04treatments vs. the placebo (T01). These results are expressed as PCV2DNA copies/ml. The results in FIG. 5 indicate that all treatments hadsignificantly less fecal shed when compared to the placebo at day 42. Inaddition, 5% Amphigen & 5% SLCD (T04) had significantly less fecal shedas compared to 5% Amphigen (T03) at day 42. No other treatmentdifferences were noted.

FIG. 6 is a graph showing the PCV2 nasal shed obtained with the T02-T04treatments vs. the placebo (T01). These results are expressed as PCV2DNA copies/ml. The results in FIG. 6 indicate that all treatments hadsignificantly less nasal shed when compared to the placebo at day 42. Inaddition, 20% SLCD (T05) had significantly less nasal shed compared to5% Amphigen (T03) at day 42. No other treatment differences were noted.

FIG. 7A and FIG. 7B are of two graphs showing the results of aninterferon-gamma (IFN-γ) test that measures PCV2-specific cellularmediated immune (CMI) responses. The CMI results are shownpost-vaccination/pre-challenge (FIG. 7A), andpost-vaccination/post-challenge (FIG. 7B). In these graphs, stimulationof 5×10⁶ cells was considered significant ( . . . ). All PCV2/M.hyoexperiment vaccines gave a detectable IFN-γ response post-vaccination.The 10% SP-oil (T02) drove the strongest IFN-γ responsepost-vaccination. The 20% SLCD (T05) induced an earlier response, butthe lowest response at day 20. There was a large post-challengeresponse, especially seen in the placebo group. Additionally, thepost-challenge response was lower in the vaccinated pig treatment groupsas compared to the placebo group.

Table 7 below shows the lymphoid depletion obtained with theexperimental treatments contrasted to the placebo.

TABLE 7 PCV2 Histopathology (Lymphoid Depletion) Lymphoid DepletionContrasted to Placebo Treatment Positive Negative % Ever Pos. P-valueSignificant Placebo 9 7 56%  NA NA 10% SP-oil 1 15 6% 0.0059 Yes 5% 1 156% 0.0059 Yes Amphigen 5% 0 16 0% 0.0008 Yes Amph + 5% SLCD 20% SLCD 115 6% 0.0059 Yes

The results presented in Table 7 above show that all vaccines affordedstrong protection against lymphoid depletion. Also, no statisticallysignificant vaccine treatment contrasts were observed. Table 8 belowshows the immunohistochemistry obtained with the experimental treatmentscontrasted to the placebo.

TABLE 8 PCV2 Histopathology (Immunohistochemistry) ImmunohistochemistryContrasted to Placebo Treatment Positive Negative % Ever Pos. P-valueSignificant Placebo 12 4 75%  NA NA 10% SP-oil 0 16 0% 0.0001 Yes 5% 115 6% 0.0002 Yes Amphigen 5% 0 16 0% 0.0001 Yes Amph + 5% SLCD 20% SLCD0 16 6% 0.0001 Yes

The results presented in Table 8 above show that all vaccines affordedstrong protection against PCV2 colonization as evidenced byimmunohistochemistry. Also, no statistically significant vaccinetreatment contrasts were observed.

In conclusion, the results presented in this example demonstrate thatthe M.hyo soluble antigen preparation does not interfere with PCV2efficacy. The results also show that all the PCV/M.hyo experimentalvaccine formulations provide efficacy against PCV2 challenge.Additionally, the results indicate that there are some statistical andnumerical differences obtained with the different adjuvant formulations,with 10% SP-oil yielding the strongest efficacy.

Example 8 Evaluation of M.hyo Efficacy of a 1-Bottle PCV2/M.hyoCombination Vaccine in with Different Adjuvant Formulations

This study was designed to evaluate the M.hyo efficacy of a 1-bottlePCV2/M.hyo combination vaccine with different adjuvant formulations. TheM.hyo antigen was combined with Porcine Circovirus (Type 1-Type 2Chimera, or PCV1-2, killed virus) in one bottle.

Processing of Fluids:

Inactivated M.hyo fermentation fluid (described above in Example 1) wastreated for each indicated group as follows.

-   T02-T04: These treatments were the same as those described for    treatment groups T02-T04 in Example 7 above.-   T05: This was formulated with inactivated M.hyo cells (M.hyo    bacterin) as described in Example 1 above under the heading    “Fermentation and Inactivation”.

All experimental PCV2/M.hyo vaccines were formulated with differentadjuvant formulations. The experimental vaccine formulations wereprepared with M.hyo antigens processed according to treatments T02-T04.In addition, Treatment T01 corresponded to a placebo (sterile saline).Treatment T05 is a positive control corresponding to an expiredRespiSure® vaccine, which is an M.hyo bacterin-based vaccine (PfizerAnimal Health).

These experimental formulations are described in Table 9 below, whereinthe symbol * indicates the M hyo antigen from global M hyo seed, ProteinA treated supernatant and the symbol ** indicates InvestigationalVeterinary Product (IVP) serial.

TABLE 9 PCV2/M. hyo Experimental Vaccine Formulations Used for M. hyoEfficacy Study in Different Adjuvant Formulations IVP PCV1-2 M Hyo*Adju- Treatment Serial ** Ag Ag vant Other T01 87-244-DK NA Sterile(Placebo) Saline T02 L0411RK08 1.6 RP 7.5 RP 10% SP Oil NA T03 L0411RK095% Amphigen T04 L0611RK03 5% Amphigen + 5% SLCD T05 A827870 Expired“RespiSure” vaccine

Pigs at 3 weeks of age were intramuscularly inoculated with a singledose of the different vaccine formulations described in Table 9 above.Fourteen animals were included in both the placebo and 10% SP-oilgroups, thirteen animals were included in the positive control group,and sixteen animals were included in both the 5% Amphigen and 5%Amphigen+5% SLCD groups.

Animals were challenged 21 days after vaccination with a virulent M.hyofield isolate. Animals were necropsied 28 days after challenge and thelungs were removed and scored for consolidation consistent with M.hyoinfection. Table 10 below contains the lung lesion scores for therespective treatment groups. Statistical significance was determined bya Mixed Model Analysis of lung scores for each group.

TABLE 10 M. hyo Lung Lesions LS Mean Lung Range % Lung Treatment #Animal Lesion Lesion Placebo (T01) 14 13.1% 0.1-50.5 10% SP-oil (T02) 14 4.3% 0.0-50.8 5% Amphigen (T03) 16  4.7% 0.0-38.5 5% Amph + 5% 16 12.0%0.1-55.8 SLCD (T04) Expired RSO (T05) 13 2.28% 0.0-34.5

As indicated in Table 10 above, the placebo group had a mean lung lesionscore of 13.1%, as compared to the 10% SP-oil and 5% Amphigen treatmentgroups which had mean lung scores of 4.3% and 4.7%, respectively. Boththe 10% SP-oil and 5% Amphigen formulations reduced and/or preventedlung lesions. Thus, the experimental PCV/M.hyo vaccines formulated with10% SP-oil or 5% Amphigen were considered efficacious. The PCV2 antigendid not appear to interfere with the M.hyo efficacy of theseformulations.

In contrast, the 5% Amphigen+5% SLCD group had a mean lung lesion scoreof 12.0%. which was an unacceptable result in that it was not differentas compared to the placebo. Consequently, the experiment PCV/M.hyovaccine formulated with 5% Amphigen+5% SLCD was not considered asefficacious.

It is noted that due to the reduced animal number and high variabilityin lung lesion scoring, no statistical treatment effect could beconclusively demonstrated in this study. For this reason, it was decidedthat another study would be designed to test the M.hyo efficacy of thePCV/M.hyo experimental formulations in 10% SP-oil. This repeat study ispresented in Example 9 below.

Example 9 Evaluation of M.hyo Efficacy of a 1-Bottle PCV2/M.hyoCombination Vaccine in 10% SP-Oil

This study is a proof of concept designed to evaluate the M.hyo fractionefficacy of four experimental PCV2/M.hyo vaccines (Serials L0711RK11,L0711RK12, L0711RK13 and L0711RK14 in Table 11 below) prepared bydifferent M.hyo manufacturing processes which utilize Protein A for IgGremoval compared to control vaccines prepared with the standard M.hyomanufacturing process. Each of these four experimental PCV2/M.hyovaccines included 10% SP-oil as the adjuvant.

Processing of Fluids:

-   T02: Inactivated M. hyopneumoniae antigen as described under    “Fermentation and Inactivation” in Example 1 above.-   T03 and T04: Formulated with inactivated M. hyopneumoniae cells as    described under “Fermentation and Inactivation” in Example 1 above.-   T05: Protein A treatment of medium used to grow M. hyopneumoniae.    PPLO (porcine heart derived) was made per manufacturer's directions    (i.e., 21 g/L) and yeast extract solution was made at 21 g/L in USP.    Yeast extract solution was added to the PPLO at 6.25% and the    mixture was sterilized by heating to 121° C. for ≧30 minutes.    Cysteine hydrochloride was prepared at 90 g/L and filter sterilized.    Dextrose solution was made by adding 450 g of dextrose per liter of    USP water followed by heat sterilization. To prepare the final    medium, porcine serum was added to the base medium at 10% followed    by cysteine at 0.01% and dextrose at 1.0%. Antibodies in the    complete PPLO media were removed by treatment with protein A.    Briefly, one liter of rProtein A Sepharose (part number 17-5199-03    GE Healthcare) was packed into a glass column (10×11.5 cm). After    removal of storage buffer, the column was treated with 2 column    volumes of 1M acetic acid. The resin was equilibrated with 5 column    volumes of 50 mM NaPO4, 1M NaCl buffer (pH 7.0). Fifteen liters of    complete PPLO medium was loaded onto the resin at a linear flow rate    of 140 cm/hour. The column flow through was collected and filter    sterilized through a 0.2 micron filter (Sartorius). The treated    medium was used propagate M. hyopneumoniae cells as described under    “Fermentation and Inactivation” above. Whole inactivated culture    (including cells) was formulated into the final vaccine.-   T06: Inactivated M. hyopneumoniae cells were prepared as described    under “Fermentation and Inactivation” in Example 1 above. The    inactivated fermentation fluid was centrifuged at ˜20,000×g (Sorvall    RCSB) for 30 min. and the supernatant was sterilized via 0.2 uM    filtration. One hundred fifteen mls of rProtein A resin (part number    12-1279-04, MAbSelect, GE Healthcare) was packed into a    chromatography column (5×6 cm). After removal of the storage buffer    and treatment with 2 column volumes of 1M acetic acid, the resin was    equilibrated with 5 column volumes of 50 mM NaPO4/1M NaCl buffer, pH    7.01. Approximately 1.2 liters of the clarified/filtered M.    hyopneumoniae antigen containing fluids were passed through the    resin at a flow rate of 120 cm/hr. The flow through was collected    and sterilized via 0.2 μM filter.-   T07: Inactivated M. hyopneumoniae cells were prepared as described    under “Fermentation and Inactivation” in Example 1 above. The    inactivated fermentation fluid was clarified by via tangential flow    filtration. Briefly, a polyether sulfone filter (GE HealthCare, part    number 56-4102-71) with nominal pore size of 0.2 μM was sanitized    with 0.5N sodium hydroxide solution followed by extensive rinsing    with sterile USP water. Inactivated mycoplasma culture fluid was    introduced to the apparatus at a recirculation rate targeted to 14.6    L/minute and a transmembrane pressure of 2-3.4 PSI. Clarification    was performed at room temperature. Filter permeate was collected and    stored at 2-8 C. until further processing. One hundred fifteen mls    of rProtein A resin (part number 12-1279-04, MAbSelect, GE    Healthcare) was packed into a chromatography column (5×6 cm). After    removal of the storage buffer and treatment with 2 column volumes of    1M acetic acid, the resin was equilibrated with 5 column volumes of    50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately 2.3 liters of the    clarified/filtered M. hyopneumoniae antigen containing fluids were    passed through the resin at a flow rate of 120 cm/hr. The flow    through was collected and sterilized via 0.2 μM filter.-   T08: Inactivated M. hyopneumoniae cells were prepared as described    under “Fermentation and Inactivation” above. The inactivated    fermentation fluid was centrifuged at ˜20,000×g (Sorvall RCSB) for    30 min. and the supernatant was sterilized via 0.2 uM filtration.    One hundred fifteen mls of rProtein A Sepharose (part number    17-5199-03 GE Healthcare) was packed into a chromatography column    (5×6 cm). After removal of the storage buffer and treatment with 2    column volumes of 1M acetic acid, the resin was equilibrated with 5    column volumes of 50 mM NaPO4/1M NaCl buffer, pH 7.01. Approximately    1.2 liters of the clarified/filtered M. hyopneumoniae antigen    containing fluids were passed through the resin at a flow rate of    120 cm/hr. The flow through was collected and sterilized via 0.2 uM    filter.

The experimental vaccine formulations were prepared with M.hyo antigensprocessed according to treatments T02-T08 above. T02, T03 and T04corresponded to positive controls. In addition, Treatment T01corresponded to a placebo (sterile saline).

These experimental formulations are described in Table 11 below. TheM.hyo antigen corresponds to the M.hyo antigen from global M.hyo seed,Protein A treated supernatant. The information in the “Protein ATreatment” column indicates whether the M.hyo supernatant was treatedwith Protein A either before or after fermentation.

TABLE 11 PCV2/M.hyo Experimental Vaccine Formulations Used for M.hyoEfficacy Study in SP-Oil Adjuvant Supernatant Treat- PCV1-2 M. hyoProtein A Clarification Protein A ment Serial No. Ag Ag Treatment MethodBrand Adjuvant Other T01 L0311AS11 NA Sterile Saline T02 A828718 NA 13Expired RespiSure One Amphigen NA T03 L0711RK09 1.5 RP 7.5 RP M.hyowithout Protein A treatment 10% SP Oil and with PCV-2 T04 L0711RK10 NAM.hyo without Protein A treatment and without PCV-2 T05 L0711RK11 1.5 RPBefore NA Sepharose T06 L0711RK12 After Centrifuge MAbSelect T07L0711RK13 After Filter MAbSelect T08 L0711RK14 After CentrifugeSepharose

Pigs at 3 weeks of age were intramuscularly inoculated with a singledose of the different vaccine formulations described in Table 11 above.There were 18 pigs included in each treatment group. Animals werechallenged 21 days after vaccination with a virulent M.hyo fieldisolate. Animals were necropsied 28 days after challenge and the lungswere removed and scored for consolidation consistent with M.hyoinfection. FIG. 8A and FIG. 8B show the lung lesion scores for therespective treatment groups. Statistical significance was determined bya Mixed Model Analysis of lung scores for each group.

The lung lesion results depicted in FIGS. 8A and 8B indicate that of allthe treatments, only two (T07 and T08) had 100% of pigs in the <5% lunglesion category. It is noted that strong statistical difference wereobserved in this study.

The results in the present example demonstrate significant M.hyoefficacy in a 1-bottle PCV2/M.hyo experimental formulation employing theProtein A-treated M.hyo supernatant and utilizing SP-oil as theadjuvant. Additionally, Example 7 above demonstrated PCV2 efficacy in a1-bottle PCV2/M.hyo formulation employing the Protein A-treated M.hyosupernatant and utilizing SP-oil as the adjuvant. Taken together, bothM.hyo and PCV2 efficacy have been demonstrated in the 1-bottlePCV2/M.hyo combinations employing Protein A-treated M.hyo supernatant.

Example 10 In Vivo Safety of Experimental PCV2/M.hyo ExperimentalVaccines

This study was conducted to evaluate in vivo safety of experimentalPCV2-M.hyo vaccines formulated at maximum antigen dose in variousadjuvant formulations in the host animal when given at the youngest age(3 weeks of age). Different adjuvant platforms were evaluated in orderto determine which of these platforms provided an acceptable safetyprofile based on temperature, injection site reactions and clinicalobservations. A 20% SLCD/10% SP-oil formulation was used as a positive(“unsafe”) control due to historic issues with injection site reactionsobserved by this investigative group and others.

Processing of Fluids:

All vaccines were prepared with inactivated M. hyopneumoniae antigen asdescribed under “Fermentation and Inactivation” in Example 1. M.hyowhole bulk antigen was used since it was known to contain soluble andinsoluble M.hyo antigens, in addition to the immunoglobulins andimmunocomplexes that would be removed upon protein A treatment. It isreasonable to conclude that removal of insoluble cell debris andimmunuoglobulins and immunocomplexes will only further enhance thesafety of the vaccine formulations. The intention of this study was tostringently test the safety of the various adjuvant formulationscontaining PCV2 antigen and M.hyo antigen. The PCV2 and M.hyo antigenswere formulated at maximum release levels to further assess safety.These experimental formulations are described in Table 12 below. IVPindicates Investigational Veterinary Product (IVP).

TABLE 12 PCV2/M. hyo Experimental Vaccine Formulations Used for SafetyStudy Minimum PCV1-2 M Hyo* Adju- Vaccine IVP Serial Ag Ag vant OtherVol. (mL) 87-244-DK NA Sterile NA (Placebo) Saline L0411RK15 7.8 RP 13RP 10% SP Oil NA 200 L0411RK16 5% Amphigen 200 L0611RK05 5% Amphigen +200 5% SLCD L0611RK06 20% SLCD + 200 10% SP Oil *M hyo antigen = fromglobal M hyo seed (whole bulk antigen).

The safety parameters employed in this study were rectal temperatureprofile and injection site reaction. The results of this study indicatedthat all candidate adjuvant platforms provided an acceptable safetyprofile in terms of rectal temperature profile and clinical observations(results not shown). Only the 20% SLCD+10% SP-oil (i.e., positivecontrol) was significantly different than the placebo vaccine and had anumber of severe injection site reactions (results not shown).

Example 11 Preparation of Protein a Treated M.hyo Antigen for PivotalStudies

FIG. 9 is a flowchart which shows one embodiment of a manufacturingprocess used to prepare PCV2 compatible Protein-A treated M.hyo antigen.Inactivated whole cultures of M.hyo were clarified of cells viatangential flow filtration. Briefly, a polyether sulfone filter (GEHealthcare, part number 56-4102-49) with nominal pore size of 0.45 μMwas sanitized with 0.5N sodium hydroxide solution followed by extensiverinsing with sterile USP water. Inactivated mycoplasma culture fluid wasintroduced to the apparatus at a recirculation rate targeted to 11.0L/minute and a transmembrane pressure of ˜5 PSI. Clarification wasperformed at room temperature. Filter permeate was collected and storedat 2-8° C. until further processing.

Following clarification, antigen containing fluids were treated withprotein A resin to reduce antibody levels. Briefly, MAbSelect protein Aresin (GE Healthcare) was packed into a glass column to a height of 12cm. The resin was equilibrated with 5 column volumes of 50 mM sodiumphosphate, 250 mM NaCl buffer (pH 7.0). Antigen containing fluid,equivalent to 10 column volumes, was loaded onto the resin at a linearflow rate of 100 cm/hour. The column flow through was collected andfilter sterilized through a 0.2 micron filter. Regeneration of thecolumn was achieved by flowing 3 column volumes of 25 mM acetatesolution at pH 3.7 followed by 4 column volumes of 1M acetic acidsolution. Anti-PCV2 antibodies and M. hyopneumoniae antigen levels weremeasured in the final antigen fluid via PCV2 specific antibody ELISA andp46 antigen quantification ELISA, respectively.

Example 12 Evaluation of Virucidal Activity Against PRRS Virus

The studies presented in this example were designed to evaluate thevarious adjuvant platforms for virucidal activity against PRRS virus.Initial experiments focused on adjuvant alone (i.e., the formulationsdid not contain PCV or M.hyo antigens). The adjuvant evaluation for PRRSvirucidal activity is presented in FIG. 10. Preliminary virucidalassessment indicated that 10% SP-oil, 0.2% Carbopol and 2.5% Amphigenare non-virucidal to PRRS virus. In contrast, the 20% SLCD adjuvantappeared to be virucidal to PRRS virus.

Further studies were performed to evaluate whether the PCV/M.hyoformulations adjuvanted with the different adjuvant platforms werenon-virucidal to PRRS virus. These results are presented in Table 13,wherein the symbol * indicates those vaccine serials which werevirucidal to PRRSV.

TABLE 13 Results of PRRS Virucidal Assay with Different FormulationsPotency Vaccine Serial Used in Studies of PCV2 PRRS Examples 7, 8, 10p46 RP NVSL Virucidal Study Description Serial # (ru/ds) RP A B ExamplesSterile Saline (0.9% 87-244-DK 7, 8, 10 Sodium chloride) (Placebo)Examples cPCV (RP 1.6) + M Hyo L0411RK08 7.1 1.29 −0.10 -0.13 7, 8 ProtA treated (RP 7.5) in 10% SP Oil Examples cPCV (RP 1.6) + M HyoL0411RK09 7.3 1.33 −0.10 +0.14 7, 8 Prot A treated (RP 7.5) in 5%Amphigen Examples cPCV (RP 1.6) + M Hyo L0611RK03 6.9 1.15 −0.36 -0.337, 8 Prot A treated (RP 7.5) in 5% Amph + 5% SLCD Example 7 cPCV (RP1.6) monovalent L0611RK04 1.50 −1.86* −0.50 in 20% SLCD Example 8Expired RespiSure One A827870 12.6 serial Example cPCV (RP 7.8) + M HyoL0411RK15 14 1.03 −0.32 −0.03 10 Whole Bulk (RP 13.3) in 10% SP OilExample cPCV (RP 7.8) + M Hyo L0411RK16 15.5 1.12 −0.36 −0.53 10 WholeBulk (RP 13.3) in 5% Amphigen Example cPCV (RP 7.8) + M Hyo L0611RK0517.5 1.50 −0.54 −0.33 10 Whole Bulk (RP 13.3) in 5% Amph + 5% SLCDExample cPCV (RP 7.8) + M Hyo L0611RK06 15.9 1.13 −1.93* −0.99* 10 WholeBulk (RP 13.3) in 20% SLCD + 10% SP Oil *Indicates Virucidal (>0.7 logloss) A—Virucidal assay control GMT ~5.53 log/mL B—Virucidal assaycontrol GMT ~6.42 log/mL

The results presented in Table 13 above indicate that 10% SP-oil isnon-virucidal to PRRS virus.

Further PCV/M.hyo vaccine serials were prepared using 10% SP-oil as theadjuvant (Table 14). The antigenic potency of these vaccine serials wascompared to a Reference PCV/M.hyo vaccine serial (L1211RK15) whichcontained 0.688% of a 20× concentrate of PCV2 antigen (prepared asdescribed in Example 2); and 9.40% of M.hyo antigen prepared asdescribed in Example 11. The results shown in Table 14 below furtherindicate that 10% SP-oil is non-virucidal to PRRS virus. The test samplevalues in Table 14 were each higher (+sign) than the virucidal assaycontrol, which had a geometric mean titer (GMT) of about 5.9±0.5 log/ml.

TABLE 14 Results of Virucidal Assay with Different PCV/ M. hyoFormulations Adjuvanted with 10% SP-oil PRRS Potency Viru- p46RP PCV2cidal Vaccine Serial (ru/ds) NVSL log10 Used Reference Reference TCID50/Description Serial # L1211RK15 L1211RK15 mL Sterile Diluent 1949122 nana (sterile water) cPCV + M Hyo L0912RK12 1.62 2.60 +0.58 Prot A treatedin 10% SP Oil cPCV + M Hyo L0912RK10 0.88 1.23 +0.58 Prot A treated in10% SP Oil cPCV + M Hyo L0912RK11 1.24 2.62 +0.58 Prot A treated in 10%SP Oil cPCV + M Hyo L0912RK08 1.08 1.03 +0.91 Prot A treated in 10% SPOil cPCV + M Hyo L0912RK09 1.65 2.06 +0.50 Prot A treated in 10% SP OilVirucidal Assay control GMT ~5.9 ± 0.5 log/ml

The results presented in this example demonstrate that 10% SP-oil isnon-virucidal to PRRS virus. The results presented in this examplefurther demonstrate that the PCV/M.hyo formulation adjuvanted with 10%SP-oil was among those vaccine serials which were considerednon-virucidal to PRRS virus (Table 13 and Table 14). In conclusion, thePCV/M.hyo formulation adjuvanted with 10% SP-oil was considered aneffective platform on which to base a trivalent combination includingPCV, M.hyo, and PRRS virus.

Example 13 Preparation of a PCV/M.hyo/PRRS Combination Vaccine

A PCV/M.hyo formulation adjuvanted with an adjuvant platform which isnon-virucidal to PRRS virus (see Tables 13 and 14 above), is provided asa ready-to-use in one-bottle liquid composition. This 1-bottle PCV/M.hyoformulation employs Protein A-treated M.hyo supernatant. Both M.hyo andPCV2 efficacy have been demonstrated in such PCV2/M.hyo formulationsemploying M.hyo Protein A-treated supernatant (see Examples 7-9). In thepresent example, this divalent PCV2/M.hyo formulation is combined with amonovalent PRRS virus antigen.

In one embodiment, a PCV/M.hyo combination in 10% SP-oil andcorresponding to one of the vaccine serials L0711RK11, L0711RK12,L0711RK13 and L0711RK14 in Table 11 above is provided as a ready-to-usein one bottle liquid composition. The results presented in Example 12above demonstrated that 10% SP-oil is non-virucidal to PRRS virus.Example 12 also demonstrated that PCV2/M.hyo formulations adjuvantedwith 10% SP-oil were among those vaccine serials which were considerednon-virucidal to PRRS virus. In the present example, such a 1-bottlePCV2/M.hyo liquid composition is used to re-hydrate a lyophilizedgenetically modified live PRRS virus composition contained in a secondbottle, such that all antigens are contained in a single bottle prior tobeing administered to a pig of a suitable age (e.g., at 3 weeks of ageor older).

In one embodiment, the PRRS virus has the genomic sequence correspondingto SEQ ID NO: 16 or a variant thereof. In another embodiment, the PRRSvirus employed in the trivalent composition is the PRRS virus isolatedesignated ISU-55, which was deposited in the ATCC under the accessionnumber VR 2430. Suitable amounts of the respective antigens aredescribed herein. Desirably, all antigens are administered in a singledose to the pig.

What is claimed is:
 1. A method for preparing an immunogenic compositionincluding a soluble portion of a Mycoplasma hyopneumoniae (M.hyo) wholecell preparation, the method comprising: i) culturing M.hyo in asuitable media over periods ranging from 18-144 hours; ii) subsequentlyinactivating the M.hyo culture; iii) harvesting the inactivated culturefluid, wherein the inactivated culture fluid comprises an M.hyo wholecell preparation comprising both a soluble liquid fraction and insolublecellular material; iv) separating the soluble liquid fraction from theinsoluble cellular material; and v) substantially removing both IgG andantigen/immunoglobulin immunocomplexes from the separated soluble liquidfraction to form a soluble portion of an M.hyo whole cell preparation.2. The method of claim 1, wherein the M.hyo culture is inactivated byexposing the culture to an inactivation agent.
 3. The method of claim 2,wherein the inactivation agent is binary ethylenimine (BEI).
 4. Themethod of claim 2, further comprising neutralizing the inactivationagent.
 5. The method of claim 1, wherein the soluble liquid fraction isseparated from the insoluble cellular material by a centrifugation step.6. The method of claim 1, wherein the soluble liquid fraction isseparated from the insoluble cellular material by a filtration step. 7.The method of claim 1, wherein the soluble liquid fraction is separatedfrom the insoluble cellular material by a precipitation step.
 8. Themethod of claim 1, wherein the soluble liquid fraction is treated with aProtein A or Protein G resin to substantially remove both the IgG andantigen/immunoglobulin immunocomplexes contained in the separatedsoluble liquid fraction.
 9. The method of claim 8, wherein the solubleliquid fraction is treated with a Protein A resin to substantiallyremove both the IgG and antigen/immunoglobulin immunocomplexes containedin the separated soluble liquid fraction.
 10. The method of claim 1,further comprising admixing the immunogenic composition with anadjuvant.
 11. The method of claim 10, wherein the adjuvant is an oilemulsion comprising a polyoxyethylene-polyoxypropylene block copolymer,squalane, polyoxyethylene sorbitan monooleate and a buffered saltsolution (SP-oil).
 12. The method of claim 1, further comprisingadmixing the immunogenic composition with a pharmaceutically-acceptablecarrier.
 13. The method of claim 1, further comprising combining theimmunogenic composition with at least one additional porcine antigen.